CN107261131A - Attenuated chimeric pig circular ring virus vaccine living - Google Patents
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Abstract
The present invention provides a kind of infectious DNA clone of new Chimeric porcine circovirus and attenuated chimeric virus living, and wherein PCV2 (being preferably hypotype PCV2b) capsid gene is incorporated into nonpathogenic PCV1 viral genomes.In one particular embodiment, PCV2 capsid genes is hypotype PCV2b, and hypotype PCV2b is the Main Subtype circulated in whole world pig.PCV1 2b attenuated chimeric virus is named as, effectively protects pig to be attacked from PCV2b and can be used as providing the live vaccine and inactivation (dead) vaccine of protection and cross protection for PCV2b and PCV2a Subtypes.The attenuated vaccine of the work of the present invention also effectively protects pig from the related disease (PCVAD) of pig circular ring virus.
Description
The application is divisional application, and the application number of its female case is：201180024467.4, international application no is PCT/
US2011/028665, the applying date is：On March 16th, 2011.
The reference of related application
The rights and interests of U.S. Provisional Patent Application No. 61/314,362 filed in 16 days March in 2010 of patent application claims, it is said
Bright book is incorporated integrally into this specification herein by reference with it.
Technical field
The present invention relates to pig circular ring virus (PCV) (particularly PCV1 and PCV2 (particularly hypotype PCV2b) chimeric disease
Poison) infectious DNA clone, the attenuated vaccine of work to inactivated vaccine and for for PCV infect it is related to pig circular ring virus
The method protected of disease (PCVAD).
Background technology
Pig circular ring virus (PCV) is a kind of small, nonencapsulated DNA virus, its belong to PCV-II section (Circoviridae)
(Todd, D. etc., 2005, Circoviridae, the 327-334 pages is loaded in (chief editors) such as C. M. Fauquet,
Virus Taxonomy: Eighth Report of the International Committee on Taxonomy of
Viruses, Elsevier Academic Press, San Diego).1 type PCV (PCV1) is in middle nineteen seventies conduct
The pollutant of pig kidney PK-15 cell lines is found (Tischer, I. etc., 1974, Characterization of
papovavirus-and picornavirus-like particles in permanent pig kidney cell
Lines (sign of papovavirus and picornavirus sample particle in permanent porcine kidney cell line), Zentralbl
Bakteriol Orig A 226:153-67)。
PCV1 is considered as nonpathogenic virus, because not drawn with the PCV1 virus inoculations pig from PK-15 cell lines
Play any disease (Tischer, I. etc., 1986, Studies on epidemiology and of pig
Pathogenicity of porcine circovirus (epidemiology and pathogenic research to pig circular ring virus),
Arch Viroi 91:271-6).In 1997, PCV variant is found that in the Canadian piggy with wasting disease
(being named as 2 type PCV (PCV2)) (Allan, G. M. etc., 1998, Isolation of porcine
circovirus-like viruses from pigs with a wasting disease in the USA and
Europe (pig circular ring virus sample virus is separated from the pig with wasting disease of US and European) J Vet Diagn
Invest 10:3-10；Clark, E. G., 1997, it is reported in pig doctors'associations of the U.S. (American Association
Of Swine Practitioners) the 28th annual meeting；Ellis, J. etc., 1998, Isolation of circovirus
From lesions of pigs with postweaning muitisystemic wasting syndrome are (from disconnected
After milk PCV-II is separated in the infringement of the pig of multisystem exhaustion syndrome), Can Vet J 39:44-51；Meehan, B.
M. etc., 1998, Characterization of novel circovirus DNAs associated with
Wasting syndromes in pigs (the new PCV-II DNA related to the exhaustion syndrome of pig sign), J Gen
Virol 79 (Pt 9):2171-9).At present, PCV2 is the main pathogens of the related disease (PCVAD) of pig circular ring virus, pig
The related disease of PCV-II is including becoming thin, dead, breathing is levied, enteritis, the scorching nephrotic syndrome of irreproducible and pigskin (PDNS)
(Opriessnig, T. etc., the associated disease of 2007, Porcine circovirus type 2:
update on current terminology, clinical manifestations, pathogenesis,
Diagnosis, and intervention strategies (the related diseases of 2 type pig circular ring virus：To current term, face
Bed performance, pathology, diagnosis and the renewal of intervention strategies), J Vet Diagn Invest 19:591-615).PCV2 quilts at present
It is considered one of most economical important viral pathogen in global swinery, and in each main man of pig producing country discovery in the world
(Gillespie, J. etc., the and porcine Circovirus- of 2009, Porcine circovirus Type 2
Associated Disease (the 2 type pig circular ring virus disease related to pig circular ring virus), J Vet Intern Med).
With in the conventional pig of the independent experimental infections of PCV2 observe severe clinical PCVAD be uncommon, and usually require with other pigs
Pathogen (such as porcine reproductive and respiratory syndrome virus (PRRSV) or pig parvoviral (PPV)) coinfection is composed entirely with inducing
Clinical PCVAD (Allan, G. M. etc., 2000, Experimental infection of colostrum
deprived piglets with porcine circovirus 2 (PCV2) and porcine reproductive
And respiratory syndrome virus (PRRSV) potentiates PCV2 replication (use pig annulus
The piggy enhancing PCV2 that 2 (PCV2) of virus and porcine reproductive and respiratory syndrome virus (PRRSV) experimental infection deprive colostrum is answered
System), Arch Virol 145:2421-9；Opriessnig, T. etc. 2007. is ibid；Roca, M. etc., 2004, In
vitro and in vivo characterization of an infectious clone of a European
Strain of porcine circovirus type 2 (infection clones external of 2 type pig circular ring virus Europe strain and
Characterize in vivo), J Gen Virol 85:1259-66；Rovira, A. etc., 2002, Experimental
inoculation of conventional pigs with porcine reproductive and respiratory
Syndrome virus and porcine circovirus2 (use porcine reproductive and respiratory syndrome virus and 2 porcine circovirus
The conventional pig of experiment inoculation), J Virol 76:3232-9；Tomas, A. etc., 2008, A meta-analysis on
Experimental infections with porcine circovirus type 2 (PCV2) are (to 2 type pig circular ring virus 2s
The comprehensive analysis of malicious (PCV2) experimental infection), Vet Microbiol 132:260-73).Cutd open however, individually being infected with PCV2
Abdomen production source deprive colostrum (CD/CD) pig caused severe clinical PCVAD and it is dead (Allan, G. etc. 2003,
Reproduction of postweaning muitisystemic wasting syndrome in pigs
The isolate of experimentally inoculated with a Swedish porcine circovirus 2 are (with auspicious
Postweaning multisystem exhaustion syndrome is reproduced in the pig of allusion quotation 2 porcine circovirus conivium experiment inoculation), J Vet Diagn
Invest 15:553-60；Allan, G. M. etc., 2004, PMWS: experimental model and co-
infections (PMWS：Experimental model and coinfection), Vet Microbiol 98:165-8；Bolin, S. R. etc.,
2001, Postweaning multisystemic wasting syndrome induced after experimental
inoculation of cesarean-derived, colostrum-deprived piglets with type 2
Porcine circovirus (induce disconnected being tested with 2 type pig circular ring virus after the piggy of colostrum is deprived in inoculation caesarean birth source
Multisystem exhaustion syndrome after milk), J Vet Diagn Invest 13:185-94；Harms, P. A. etc., 2001,
Experimental reproduction of severe disease in CD/CD pigs concurrently
infected with type 2 porcine circovirus and porcine reproductive and
Respiratory syndrome virus (infect simultaneously with 2 type pig circular ring virus and porcine reproductive and respiratory syndrome virus
CD/CD pigs in experiment reproduce severe disease), Vet Pathol 38:528-39；Kennedy, S. etc., 2000,
Reproduction of lesions of postweaning muitisystemic wasting syndrome by
infection of conventional pigs with porcine circovirus type 2 alone or in
Combination with porcine parvovirus (individually or with pig parvoviral combine by using 2 type pig circular ring virus
Conventional pig is infected to reproduce the infringement of postweaning multisystem exhaustion syndrome), J Comp Pathol 122:9-24).PCV2's
Several pathology, immunology and molecular biology integrate summary be it is available (Allan, G. M. and J. A. Ellis,
2000, Porcine circoviruses:A review (pig circular ring virus：Summary) J Vet Diagn Invest 12:
3-14；Ellis, J. etc., 2004, Porcine circovirus-2 and concurrent infections in
The field (pig circular ring virus -2 and field accompanying infection), Vet Microbiol 98:159-63；Finsterbusch,
T. with A. Mankertz, 2009, Porcine circoviruses-small but powerful (pig circular ring virus-
It is small but strong), Virus Res 143:177-83；Gillespie, J. etc., 2009, ibid；Mankertz, A. etc.,
2004, Molecular biology of Porcine circovirus: analyses of gene expression
The and viral replication (molecular biology of pig circular ring virus：Gene expression and the analysis of virus replication), Vet
Microbiol 98:81-8；Opriessnig, T. etc. 2007, ibid；Ramamoorthy, S. and X. J. Meng,
2009, Porcine circoviruses:A minuscule yet mammoth paradox (pig circular ring virus：One micro-
Small but huge antinomy), Anim Health Res Rev 10:1-20；Segales, J. etc., 2005, porcine
Circovirus diseases (pig circular ring virus disease), Anim Health Res Rev 6:119-42).
Although pathogenic PCV2 is similar with nonpathogenic PCV1 genome organization, PCV1 and PCV2 genome are enjoyed
There are only about 68-76% nucleotide sequence homology (Fenaux, M. etc., 2004, Detection and in vitro
and in vivo characterization of porcine circovirus DNA from a porcine-derived
Commercial pepsin product (from pig originate business pepsin product pig circular ring virus DNA detection with
And in vitro and in vivo is characterized), J Gen Virol 85:3377-82；Hamel, A. L etc., 1998, Nucleotide
sequence of porcine circovirus associated with postweaning muitisystemic
The wasting syndrome in pigs (nucleotides of the pig circular ring virus relevant with the postweaning multisystem exhaustion syndrome of pig
Sequence), J Virol 72:5262-7；Tischer, I. etc., 1982, A very small porcine virus
With circular single-stranded DNA (the very small swine disease poison with circular single stranded DNA), Nature
295:64-6), and difference (Cheung, the A. K. in the antigen overview of transcriptional profile and capsid protein is had reported
2003, Comparative analysis of the transcriptional patterns of pathogenic and
The nonpathogenic porcine circoviruses (comparisons point caused a disease with the transcriptional profile of nonpathogenic pig circular ring virus
Analysis), Virology 310:41-9；Lekcharoensuk, P. etc., 2004, Epitope mapping of the
major capsid proteins of type 2 porcine circovirus (PCV2) by using chimeric
PCV1 and PCV2 (are entered by using chimeric PCV1 and PCV2 come the major capsid protein to 2 type pig circular ring virus (PCV2)
Row epitope mapping), J Virol 78:8135-45；Shang, S. B. etc., 2009, Fine mapping of
antigenic epitopes on capsid protein of porcine circovirus, and antigenic
Phenotype of porcine circovirus type 2 (epitope on the capsid protein of pig circular ring virus it is excellent
Good mapping and the antigenic phenotype of 2 type pig circular ring virus), Mol Immunol 46:327-34).By the two of viral genome codes
Individual oligogene include 942 bp replicase (rep) genes (Mankertz, A. and B. Hillenbrand, 2001,
Replication of porcine circovirus type 1 requires two proteins encoded by the
Viral rep gene (duplication of 1 type pig circular ring virus needs two kinds of albumen by viral rep gene codes), Virology
279:429-38) with 702 bp capsid genes (cap) (Nawagitgul, P. etc., 2000, Open reading frame
The encodes a major capsid protein of 2 of porcine circovirus type 2 be (2 type pig circular ring virus
ORFs 2 encodes a kind of main capsid protein), J Gen Virol 81:2281-7).Rep genes PCV1 with
It is highly conserved between PCV2, with about 83% nucleotide sequence homology, and cap genes enjoy the same of only about 67-70%
One property (Mankertz, A. etc., 2004, ibid).At present, at least three kinds of PCV2 Asia has been identified in whole world swinery
Type：PCV2a, PCV2b and PCV2c (Dupont, K. etc., 2008, Genomic analysis of PCV2 isolates
from Danish archives and a current PMWS case-control study supports a shift
The in genotypes with time (genome analysises from Denmark's archives and the PCV2 coniviums of current PMWS cases-right
Genotype is supported to change with the time according to research), Vet Microbiol 128:56-64；Segales, J. etc., 2008,
PCV-2 genotype definition and nomenclature (PCV-2 VDA genotypes and name), Vet Rec
162:867-8).PCV2a and PCV2b it is both related from the clinical PCVAD of the different orders of severity (An, D. J. etc.,
2007, Phylogenetic characterization of porcine circovirus type 2 in PMWS and
The and 2006 of PDNS Korean pigs between 1999 (2 of PMWS and PDNS South Korea pig between 1999-2006
The systematic growth of type pig circular ring virus is characterized), Virus Res 129:115-22；Ciacci-Zanella, J. R. etc.,
2009, Detection of Porcine Circovirus type 2 (PCV2) variants PCV2-1 and PCV2-
2 in Brazilian pig population (2 type pig circular ring virus (PCV2) the variant PCV2-1 and PCV2-2 of Brazilian swinery
Detection), Res Vet Sci 87:157-60；Lager, K. M etc., 2007, Mortality in pigs given
The viruses derived from DNA clones of 1 and of porcine circovirus 2 subgroup of type 2 (give
Give the death rate from the viral pig of the 2 type pig circular ring virus hypotype 1 and 2 of DNA clone), Vet Rec 161:428-9；
Madson, D. M. etc., 2008, Characterization of shedding patterns of Porcine
Circovirus types 2a and 2b in experimentally inoculated mature boars (experiment inoculations
Ripe wild boar in 2a and 2b type pig circular ring virus release mode characterize), J Vet Diagn invest 20:725-
34；Opriessnig, T. etc., 2006, Genetic and experimental comparison of porcine
circovirus type 2 (PCV2) isolates from cases with and without PCV2-associated
Lesions provides evidence for differences in virulence (come from and damaged with and without PCV2 correlations
The gene of 2 type pig circular ring virus (PCV2) coniviums of harmful case and experiment are compared provides evidence for Virulence Difference), J Gen
Virol 87:2923-32；Opriessnig, T. etc., 2008, Differences in virulence among
porcine circovirus type 2 isolates are unrelated to cluster type 2a or 2b and
The prior infection provides heterologous protection (poison between 2 type pig circular ring virus coniviums
Power difference it is uncorrelated to cluster type 2a or 2b and infection before provide xenogenesis protection), J Gen Virol 89:2482-91).
Before 2005, PCV2a is only found in the swinery of America ＆ Canada, and there is PCV2a and PCV2b two in Europe and China
Person (Chae, J. S. and K. S. Choi, 2009, Genetic diversity of porcine circovirus
The from pigs in Republic of Korea (genetic diversities of the 2 type pig circular ring virus of the pig from South Korea of type 2
Property), Res Vet Sci；Dupont, K. etc., 2008, ibid).Since two thousand five, new PCV2b strains are in U.S.'s quilt
Identification, and the global change present in swinery is the dominant prevailing of PCV2b, while clinic PCVAD seriousness increase
(Carman, S. etc., 2008, The emergence of a new strain of porcine circovirus-2
in Ontario and Quebec swine and its association with severe porcine
The circovirus associated disease-2004-2006 (pig circular ring virus -2 occurred in Ontario and Quebec pig
New strain and its disease related to severe pig circular ring virus relation -2004-2006), Can J Vet Res 72:
259-68；Chae, J. S. and K. S. Choi, 2009, ibid；Cheung, A. K. etc., 2007, Detection
of two porcine circovirus type 2 genotypic groups in United States swine
Herds (type that two kind of 2 type pig circular ring virus genotype is detected in U.S. swinery), Arch Virol 152:1035-
44；Ciacci-Zanella, J. R. etc., 2009, ibid；Dupont, K. etc., 2008, ibid；Gagnon, C.
A. etc., 2007, The emergence of porcine circovirus 2b genotype (PCV-2b) in
(there is 2 porcine circovirus b gene type (PCV-2b)) in Canadian pig in swine in Canada, Can Vet J 48:
811-9；Lipej, Z. etc., 2005, Postweaning muitisystemic wasting syndrome (PMWS) in
pigs in Croatia: detection and characterisation of porcine circovirus type 2
(PCV2) (the postweaning multisystem exhaustion syndrome (PMWS) of Croatia pig：2 type pig circular ring virus of detection and sign
(PCV2)), Acta Vet Hung 53:385-96；Wang, F. etc., 2009, Genetic variation
(the China of 2 type pig circular ring virus of analysis of Chinese strains of porcine circovirus type 2
The Genetic Variation Analysis of strain), Virus Res 145:151-6；Wiederkehr, D. D. etc., 2009, A new
emerging genotype subgroup within PCV-2b dominates the PMWS epizooty in
Switzerland (the PMWS diseases of emerging genotype hypotype control Switzerland in PCV-2b), Vet Microbiol 136:
27-35).PCV2c's is pathogenic unclear because it only in 1980,1987 and nineteen ninety in Denmark without disease swinery in quilt
Report (Dupont, K. etc., 2008, ibid).
Current available commercial vaccine is all the killed vaccine based on PCV2a hypotypes or recombinant vaccine (Opriessnig, T.
Deng 2007, ibid；Ramamoorthy, S. and X. J. Meng, 2009, ibid).Successfully developed before inventor
Inactivated vaccine Suvaxyn based on PCV1-2a embedded viruses (capsid gene in PCV1 skeleton with PCV2a)
PCV2® One dose™(Fenaux, M. etc., 2004A, A chimetic porcine circovirus (PCV)
with the immunogenic capsid gene of the pathogenic PCV type 2 (PCV2) cloned
into the genomic backbone of the nonpathogenic PCV1 induces protective
Immunity against PCV2 infection in pigs (the immunogenicity capsid genes of 2 pathogenic type PCV (PCV2)
The guarantor that Chimeric porcine circovirus (PCV) induction being cloned into nonpathogenic PCV1 genome skeleton is infected for the PCV2 of pig
Shield property is immune), J Virol 78:6297-303；Fenaux, M. etc., 2003, Immunogenicity and
pathogenicity of chimeric infectious DNA clones of pathogenic porcine
Circovirus type 2 (PCV2) and nonpathogenic PCV1 in weanling pigs (wean pig cause a disease
2 type pig circular ring virus (PCV2) and the nonpathogenic PCV1 immunogenicity of chimeric infectious DNA clone and pathogenic), J
Virol 77:11232-43；Gillespie, J. etc., 2008, A genetically engineered chimeric
vaccine against porcine circovirus type 2 (PCV2) is genetically stable in
Vitro and in vivo are (hereditary in vitro and in vivo steady for the genetic engineering chimeric of 2 type pig circular ring virus (PCV2)
It is fixed), Vaccine 26:4231-6).However, because PCV2b hypotypes turn into and severe clinical PCVAD in market pig now
Related global main genotypes, and because PCV2a and PCV2b has the difference of up to 10% nucleotide sequence homology
(Fenaux, M. etc., the porcine circovirus of 2000, Genetic characterization of type 2
(PCV-2) from pigs with postweaning muitisystemic wasting syndrome in
different geographic regions of North America and development of a
differential PCR-restriction fragment length polymorphism assay to detect and
(genetic characterization comes from North America differently to differentiate between infections with PCV-1 and PCV-2
Manage the 2 type pig circular ring virus (PCV-2) and exploitation difference PCR- limits of the pig for suffering from postweaning multisystem exhaustion syndrome in region
Property fragment length polymorphism processed determines to detect and distinguish the infection between PCV-1 and PCV-2), J Clin Microbiol
38:2494-503；Olvera, A. etc., 2007, Molecular evolution of porcine circovirus
type 2 genomes:The phylogeny and clonality (molecular evolutions of 2 type pig circular ring virus genomes：System is sent out
Educate and clonality), Virology 357:175-85), so whether being currently based on the killed vaccine or again of PCV2a hypotypes
It is unknown that group vaccine, which is directed to the PCV2b hypotypes completely newly recognized and provides protection comprehensively,.Some researchs are proved presently commercially available vaccine
Validity (Fort, the M. etc., 2008, Porcine circovirus type 2 (PCV2) attacked for PCV2b
vaccination of conventional pigs prevents viremia against PCV2 isolates of
Different genotypes and geographic origins (2 type pig circular ring virus (PCV2) vaccine inoculations routine pigs
PCV2 coniviums for different genotype and geographic origin prevent viremia virusemia), Vaccine 26:1063-71；Fort,
M. etc., (PCV2) the sub-unit vaccine of 2009, One dose of a porcine circovirus 2
administered to 3-week-old conventional piglets elicits cell-mediated
Immunity and significantly reduces PCV2 viremia in an experimental model (will be single
2 porcine circovirus (PCV2) subunit vaccine of dosage gives the conventional piggy of 3 week old and causes cell-mediated immune and significantly drop
PCV2 viremia virusemias in low experimental model), Vaccine 27:4031-7；Opriessnig, T. etc., 2009,
Comparison of efficacy of commercial one dose and two dose PCV2 vaccines
using a mixed PRRSV-PCV2-SIV clinical infection model 2-3-months post
(the 2-3 months compare business single dose using the PRRSV-PCV2-SIV clinical infections model of mixing to vaccination after vaccine inoculation
Effect of amount and dose double PCV2 vaccines), Vaccine 27:1002-7), however exploitation is sub- based on PCV2b for PCVAD
The vaccine of type is necessary, and preferably live attenuated vaccine.Work attenuated vaccine based on new PCV2b hypotypes may compare in this area
Currently available killed vaccine and subunit vaccine based on PCV2a hypotypes provide bigger protection.
The content of the invention
The present invention provides a kind of nucleic acid molecules of pig circular ring virus (PCV), and it includes the nucleic acid point for encoding nonpathogenic chimeric PCV
Son, the chimeric PCV (is preferably hypotype from 1 type PCV (PCV1) genome sequence and 2 type PCV (PCV2)
PCV2b at least a portion coded sequence of capsid protein).
In one embodiment of the invention, the coded sequence of PCV2 capsid protein is selected from hypotype PCV2a, PCV2b
And PCV2c.
In another embodiment of the present invention, the coded sequence of PCV2 capsid protein is hypotype PCV2b's.
In another embodiment of the present invention, the coded sequence of PCV2 capsid protein is that PCV2 (is preferably hypotype
PCV2b at least a portion ORFs 2 (ORF2)).
In yet another embodiment of the present invention, nucleic acid molecule encoding exceedes the nonpathogenic chimeric PCV of copy,
Genome sequences and 2 type PCVs (PCV2) (be preferably hypotype PCV2b) of the chimeric PCV from 1 type PCV (PCV1)
At least a portion coded sequence of capsid protein.
The present invention also provides a kind of biological function plasmid or viral vector, and it includes the core for encoding nonpathogenic chimeric PCV
Acid molecule, the chimeric PCV (is preferably hypotype from 1 type PCV (PCV1) genome sequence and 2 type PCV (PCV2)
PCV2b at least a portion coded sequence of capsid protein).
The present invention also provides a kind of Suitable host cells transfected by carrier, and the carrier is nonpathogenic chimeric comprising encoding
PCV nucleic acid molecules, the chimeric PCV derives from 1 type PCV (PCV1) genome sequence and 2 type PCV (PCV2) (preferably
For hypotype PCV2b) capsid protein at least a portion coded sequence.
The present invention also provides a kind of avirulent infection sex-mosaicism PCV produced by Suitable host cells, the host cell quilt
Carrier transfection comprising the nucleic acid molecules for encoding nonpathogenic chimeric PCV, the chimeric PCV is from 1 type PCV's (PCV1)
At least a portion coded sequence of genome sequence and 2 type PCV (PCV2) (being preferably hypotype PCV2b) capsid protein.
The present invention also provides a kind of chimeric PCV of inactivation, and it includes 2 type PCV's (PCV2) (being preferably hypotype PCV2b)
At least a portion of capsid protein.
In one embodiment of the invention, the coded sequence of PCV2 capsid protein is selected from hypotype PCV2a, PCV2b
And PCV2c.
In another embodiment of the present invention, the coded sequence of PCV2 capsid protein is hypotype PCV2b's.
In another embodiment of the present invention, the coded sequence of PCV2 capsid protein is PCV2 at least a portion
ORFs 2 (ORF2).
The present invention also provides a kind of viral vaccine, its comprising physiologically acceptable carrier and immunogenicity amount be selected from
Under member：(a) nucleic acid molecules of pig circular ring virus (PCV), it includes the nonpathogenic PCV of encoding chimera nucleic acid molecules, institute
State chimeric nonpathogenic PCV from 1 type PCV (PCV1) genome sequence and 2 type PCV (PCV2) capsid protein extremely
Few a part of coded sequence, (b) biological function plasmid or viral vector, it includes the core containing the nonpathogenic PCV of encoding chimera
The PCV of acid molecule nucleic acid molecules, it is described to be fitted together to nonpathogenic PCV from PCV1 genome sequence and PCV2 capsid egg
White at least a portion coded sequence, the nonpathogenic chimeric PCV of (c) avirulent infection, it includes non-pathogenic containing encoding chimera
PCV nucleic acid molecules PCV nucleic acid molecules, it is described be fitted together to nonpathogenic PCV from PCV1 genome sequence and
At least a portion coded sequence of PCV2 capsid protein, and the chimeric PCV that (d) is inactivated, it (is preferably hypotype comprising PCV2
PCV2b at least a portion of capsid protein).
In one embodiment of the invention, vaccine includes chimeric PCV viruses living.
In one embodiment of the invention, chimeric PCV virus of the vaccine comprising inactivation.
In another embodiment of the present invention, vaccine additionally comprises adjuvant.
In another embodiment of the present invention, vaccine is for PCV2a and PCV2b infection protections.
The present invention also provides a kind of method for the viral infection immunity pigs of PCV2, and it is included the virus of immune effective dose
Vaccine gives pig, and the viral vaccine is selected from following member comprising physiologically acceptable carrier and immunogenicity amount：(a)
The nucleic acid molecules of pig circular ring virus (PCV), it includes the nonpathogenic PCV of encoding chimera nucleic acid molecules, described chimeric non-pathogenic
PCV from 1 type PCV (PCV1) genome sequence and 2 type PCV (PCV2) capsid protein at least a portion coding
Sequence, (b) biological function plasmid or viral vector, it includes the PCV of the nucleic acid molecules containing the nonpathogenic PCV of encoding chimera
Nucleic acid molecules, it is described be fitted together to nonpathogenic PCV from PCV1 genome sequence and PCV2 capsid protein at least one
Partial coding sequence, the nonpathogenic chimeric PCV of (c) avirulent infection, it includes the core containing the nonpathogenic PCV of encoding chimera
The PCV of acid molecule nucleic acid molecules, it is described to be fitted together to nonpathogenic PCV from PCV1 genome sequence and PCV2 capsid egg
White at least a portion coded sequence, and the chimeric PCV that (d) is inactivated, it includes the capsid of PCV2 (being preferably hypotype PCV2b)
At least a portion of albumen.
In one embodiment of the invention, methods described is included nucleic acid molecules or the attenuated chimeric PCV of work viruses
Give pig.
In one embodiment of the invention, methods described includes giving pig by the chimeric PCV viruses of inactivation.
In another embodiment of the present invention, methods described include will be parenteral, intranasal, intracutaneous or percutaneous by vaccine
Give pig.
In yet another embodiment of the present invention, methods described includes that in vaccine lymph or intramuscular pig will be given.
The present invention also provides a kind of method that disease (PCVAD) related for pig circular ring virus protects pig, and it includes will
The viral vaccine of immune effective dose gives pig, choosing of the viral vaccine comprising physiologically acceptable carrier and immunogenicity amount
From following member：(a) nucleic acid molecules of pig circular ring virus (PCV), its nucleic acid comprising the nonpathogenic PCV of encoding chimera point
Son, it is described to be fitted together to nonpathogenic PCV from 1 type PCV (PCV1) genome sequence and 2 type PCV (PCV2) capsid egg
White at least a portion coded sequence, (b) biological function plasmid or viral vector, it includes nonpathogenic containing encoding chimera
The PCV of PCV nucleic acid molecules nucleic acid molecules, it is described to be fitted together to genome sequence and PCV2 that nonpathogenic PCV derives from PCV1
Capsid protein at least a portion coded sequence, the nonpathogenic chimeric PCV of (c) avirulent infection, its include containing encode it is embedding
The PCV of nonpathogenic PCV nucleic acid molecules nucleic acid molecules are closed, it is described to be fitted together to the genome that nonpathogenic PCV derives from PCV1
At least a portion coded sequence of sequence and PCV2 capsid protein, and the chimeric PCV that (d) is inactivated, it includes PCV2 (preferably
For hypotype PCV2b) capsid protein at least a portion.
Brief description of the drawings
Following detailed description of the invention are read in conjunction with the figure, the features described above of the present invention can be more clearly understood that, wherein：
Fig. 1 illustrates the structure and genome organization of total length PCV2b and chimeric PCV1-2b DNA clones.Fig. 1 (A) shows that PCV2b is mono-
Body DNA clone.With PCR primer A (SEQ ID No:3) with B (SEQ ID No:4) after (table 1) amplification, uniqueness is used
Sac II sites, the clone wild-type PCV2b complete genome group in pBSK+ carriers.The chimeric PCV1-2b of Fig. 1 (B) displays is mono-
Body DNA clone.Use PCR primer C-H (SEQ ID No:5-10) (table 1), is built chimeric by Overlap extension PCR
PCV1-2b DNA clones.
Fig. 2 illustrates the caesarean birth source being inoculated with chimeric PCV1-2b viruses and wild type PCV2b Viral experiments and deprives colostrum
(CD/CD) pig in PCV2 capsids specific antibody reaction.Average value serum S/P ratios are drawn to each treatment group through experiment
+/- SEM, wherein (*) represents dramatically different in that day.Due to dead or some PCV2b infection that is euthanized in early days pig, in 35-
The pig sampled in 42 dpi group is less than 5.With different alphabetical groups in that day to be dramatically different.
Fig. 3 shows the caesarean birth source stripping with PBS, chimeric PCV1-2b viruses and wild type PCV2b virus inoculations
Take comparison of the overall lymph infringement scoring in (CD/CD) pig of colostrum in 21 dpi by force.Compare processing between lymph node, spleen and
Lymph atrophy (lymphoid depletion), histocyte displacement and the scoring of PCV2 specific antigens of amygdaline combination.
Include in this analysis in the pig of PCV2b inoculations dead 18 dpi.Circle represents median, and case is the 1st and the 3rd quartile
Number, and case must the whole data areas of (whisker) expression.It is dramatically different with different alphabetical processing.
Fig. 4 illustrates the (CD/ that colostrum is deprived in the caesarean birth source being inoculated with chimeric PCV1-2b viruses and PCV2b Viral experiments
CD) viral DNA carrying capacity in the serum and lymphoid tissue of pig is quantified.Fig. 4 (A) displays quantify the disease in serum using qPCR
Toxaemia and viral DNA carrying capacity.The cell mean +/- SEM of log viral genome copies per ml serum is drawn to each treatment group,
Wherein (*) represents dramatically different in that day.0 dpi all samples and from PBS be inoculated with pig all samples for feminine gender.
Due to dead or some PCV2b infection that is euthanized in early days pig, the pig sampled in 35-42 dpi group is less than 5.Fig. 4 (B)
Display quantifies the viral DNA carrying capacity in TBLN tissues using qPCR.Drawn in that day that pig is dead or is euthanized to each pig
The lymph node tissue viral DNA carrying capacity determined, wherein drawing the +/- SEM of cell mean in 21 and 42 dpi.From PBS inoculations
The all samples of pig be feminine gender, or the pig (solid circles) of PCV2b infection that is in early days euthanized dead due to PCVAD is no
It is included in the analysis of cell mean.(*) represents dramatically different in that day.
Fig. 5 is illustrated with chimeric PCV1-2b viral vaccinations and then with PCV2a or PCV2b Wild-Type Virus Challenges
The conventional PCV2 specific antibodies reaction without in specific pathogen pig.Average value serum S/ is drawn to each treatment group through experiment
P is than +/- SEM, wherein (*) represents dramatically different between vaccine inoculation group (vax) and non-vaccine inoculation group (PBS).Use PCV2a
Or PCV2b attacks all pigs in 56 dpv.77 dpv's (or 21 dpc) there are different alphabetical groups to be dramatically different in that day
's.
The conventional SPF that Fig. 6 displays are inoculated with PCV1-2b vaccines or PBS and then attacked with PCV2a or PCV2b
The comparison of overall lymph infringement scoring in pig.By attacking PCV2 virus subtypes, compare vaccine inoculation pig and non-vaccine inoculation
Lymph atrophy, histocyte displacement and the scoring of PCV2 specific antigens of lymph node, spleen and amygdaline combination between pig.Circle
Circle represents median, and case is the 1st and the 3rd quartile, and case must represent data area.Processing with (*) is not to for significantly
Together.
Fig. 7 is illustrated with PBS or PCV1-2b vaccine inoculations and then with the PCV2a or PCV2b conventional pigs attacked
The detection of PCV2 viral DNA carrying capacity in serum and lymphoid tissue sample and quantitative.Fig. 7 (A) displays quantify blood using qPCR
Viremia virusemia and virus load in clear.The cell mean log viral genome copies drawn to each treatment group per ml serum are +/-
SEM.56 dpv (or 0 dpc) all samples and all samples from vaccine inoculation pig are to PCV2a or PCV2b DNA
For feminine gender.Fig. 7 (B) displays quantify the viral DNA carrying capacity in TBLN tissues using qPCR.Draw the lymph determined to each pig
Median viral DNA carrying capacity in tissue.Circle represents median, and case is the 1st and the 3rd quartile, and case must be represented all
Data area.When with PCV2a or PCV2b attacks, virus is detected in the lymph node of the pig of all non-vaccine inoculations
DNA, but only 1/10 vax/PCV2b pigs and 5/10 vax/PCV2a pigs have detectable viral DNA.(*) represents to be based on
Challenge virus hypotype, it is dramatically different between vaccine inoculation pair and non-vaccine inoculation pair.
Detailed description of the invention
PCV2 infects and PCVAD persistently forms chief threat to global swinery.PCVAD is probably most passing through of facing of nowadays pig industry
Important disease in Ji.Become thin, lymph atrophy and histocyte infiltration micro- infringement and infringement in exist PCV2 antigens or
DNA is three characteristic standards (Segales, J. etc., 2005, ibid) for the PCVAD for diagnosing pig.However, it is reported that not being institute
Clinic PCVAD will occur for the pig for having infection PCV2, and coinfection virus and bacterial pathogens are usual to the clinical PCVAD for inducing full spectrum
It is required (Albina, E. etc., 2001, An experimental model for post-weaning
Muitisystemic wasting syndrome (PMWS) in growing piglets are (after a kind of wean of development piggy
The experimental model of multisystem exhaustion syndrome (PMWS)), J Comp Pathol 125:292-303；Magar, R. etc.,
2000, Experimental transmission of porcine circovirus type 2 (PCV2) in weaned
pigs:A sequential study (the experiment transmission of the 2 type pig circular ring virus (PCV2) of wean pig：Sequential research), J
Comp Pathol 123:258-69).Before 2005, that is detected from the PCVAD cases of America ＆ Canada is viral several
It is exclusively PCV2a hypotypes.Therefore, four kinds of commercial vaccines for being all based on PCV2a hypotypes are developed and at present in the whole world
Used in swinery.The PCV2a strains of whole world separation are nucleotide sequence homologies that is closely related and enjoying 95-99%, and because
This these commercial vaccine is effectively for the PCV2a and PCVAD in global swinery.
Recently, the more serious PCVAD cases of the certain areas outburst of America ＆ Canada have been attributed to the one kind occurred
New PCV2b hypotypes (Gagnon, C. A. etc., 2007, ibid).Nucleotide sequence phase between PCV2a and PCV2b hypotypes
Difference up to 10%, and identified distinguish two kinds of hypotypes unique amino acid sequence motif (Cheung, A. K. etc., 2007, together
On), so that it is complete to propose that the commercial vaccine for being based entirely on PCV2a hypotypes whether at present can be carried out for new PCV2b Subtypes
The problem of face is protected.In recent years, the PCV2 of global swinery is prevailing to be mainly transferred to PCV2b hypotypes, in fact, the U.S. is big
Most nearest PCVAD cases it is related to new PCV2b hypotypes (Cheung, A. K. etc., 2007, ibid；Firth, C
Deng 2009, Insights into the evolutionary history of an emerging livestock
pathogen:Porcine circovirus 2 (see clearly the evolutionary history of emerging domestic animal pathogen：2 porcine circovirus), J
Virol 83:12813-21).Vaccine based on PCV2a hypotypes is still used in the whole world, because PCV2a vaccines have shown offer
Cross protection (Fort, M. etc., 2008, ibid；Fort, M. etc. 2009, ibid；Opriessnig, T. etc.,
2009, ibid；Segales, J. etc., 2008, ibid).However, the vaccine based on PCV2a provided for circulation
The cross protection degree of new PCV2b hypotypes is unknown, and global pig industry must benefit from use based on circulation at present
Main PCV2b hypotypes vaccine.Therefore, an object of the present invention is to develop a kind of based on the new of new PCV2b hypotypes
Generation vaccine simultaneously evaluates the vaccine based on PCV2b for both effects of PCV2a and PCV2b attacks.
The present invention provides a kind of PCV1 and PCV2 of the work of attenuation embedded virus.In one particular embodiment, structure
The PCV1 viruses of expression PCV2 hypotypes PCV2b capsid protein are built.Illustratively property method, inventor generates first
The infectious DNA clone of PCV2b hypotypes, then constructs new embedded virus PCV1-2b, its gene in nonpathogenic PCV1
The immunogenicity capsid gene of PCV2b hypotypes is included in group skeleton.New chimeric PCV1-2b diseases are evaluated in CD/CD pigs first
The pathogenic and immunogenicity of poison.Then, attacked in conventional pig and intersect Attack Research to determine that PCV1-2b is fitted together to epidemic disease
The efficacy of vaccines of seedling diseases poison.Inventor confirms that chimeric PCV1-2b vaccine viruses are to be attenuated and induce to be directed to PCV2b in pig
Protective immunity and the cross-protective immunity for PCV2a.Therefore, the new chimeric PCV1-2b vaccines should be conduct
For both PCV2b and PCV2a infection and the excellent candidates of the attenuated vaccine of PCVAD work.
Following embodiments show certain aspects of the invention.It is to be appreciated, however, that these embodiments are said merely to illustrating
It is bright, it is not intended to be entirely limited the condition and scope of the present invention.Typical reaction condition is provided (for example, temperature it should be appreciated that working as
Degree, reaction time etc.) when, although generally more undesirable, but the condition above and below particular range can be used.In room temperature
Embodiment is carried out under (about 23 DEG C-about 28 DEG C) and atmospheric pressure.Unless otherwise, otherwise present document relates to all numbers and percentage
Than based on weight, all temperature are degree Celsius to represent.
Embodiment 1
The viral coniviums of PCV1 and PCV2：
The infectious DNA clones of PCV1 are constructed in research before and nonpathogenic (Fenaux, M. are shown as in pig
Deng the porcine circovirus is infectious when of 2002, Cloned genomic DNA of type 2
injected directly into the liver and lymph nodes of pigs: characterization of
Clinical disease, virus distribution, and pathologic lesions are (when being injected directly into pig
When in liver and lymph node, the genomic DNA of the clone of 2 type pig circular ring virus is infective：Clinical disease, viral distribu-tion and disease
Manage the sign of infringement), J Virol 76:541-51；The 2004A such as Fenaux, M., ibid；Fenaux, M. etc.,
2003, ibid).PCV2a coniviums ISU-40895 (SEQ ID No:1, Genbank accession number AF264042) in 1998 years
Found in the pig with PCVAD on Iowa farm (Fenaux, M. etc., 2000, ibid), and be widely used in
PCV2 Study on Pathogenicity (Fenaux, M. etc., 2002, ibid；Fenaux, M. etc., 2004, ibid；Fenaux, M.
Deng, 2003, ibid；Opriessnig, T. etc., 2006, Evidence of breed-dependent
differences in susceptibility to porcine circovirus type-2-associated disease
And lesions (evidence for being susceptible to suffer from the kind dependence sex differernce of the related disease of 2 type pig circular ring virus and infringement), Vet
Pathol 43:281-93；Opriessnig, T. etc., 2006, Effects of the timing of the
administration of Mycoplasma hyopneumoniae bacterin on the development of
(the delivery times of Mycoplasma hyopneumoniae bacterin of lesions associated with porcine circovirus type 2
To occurring the influence of the infringement related to 2 type pig circular ring virus), Vet Rec 158:149-54；Opriessnig, T. etc.,
2004, Experimental reproduction of postweaning muitisystemic wasting syndrome
in pigs by dual infection with Mycoplasma hyopneumoniae and porcine
Circovirus type 2 (are tested by using mycoplasma hyopneumoniae and 2 type pig circular ring virus double infections and are reproduced the disconnected of pig
Multisystem exhaustion syndrome after milk), Vet Pathol 41:624-40；Opriessnig, T. etc., 2003, Effect
of vaccination with selective bacterins on conventional pigs infected with
The porcine circovirus of type 2 are (with shadow of the selective bacterination to the conventional pig with 2 type Infection of Porcine circovirus
Ring), Vet Pathol 40:521-9).PCV2a-40895 can cause the micro- infringements of PCVAD and clinic under experimental conditions
Disease (Opriessnig, T. etc., 2006, ibid；Opriessnig, T. etc., 2004, Effect of porcine
parvovirus vaccination on the development of PMWS in segregated early weaned
(pig is tiny by the porcine circovirus and porcine parvovirus of pigs coinfected with type 2
Viral vaccination in the isolation early weaning pig with 2 type pig circular ring virus and pig parvoviral coinfection to occurring PMWS's
Influence), Vet Microbiol 98:209-20；Opriessnig, T. etc., 2004, ibid).Complete by sequencing
Viral genome (SEQ ID No:2, Genbank accession number GU799576), it was confirmed that the PCV2b strains used in research are true
Positive PCV2b hypotypes.PCV2b genomic DNA is used as infectious DNA clone and chimeric PCV1- for building PCV2b
The source of 2b infectivity DNA clones.Before making the present invention, the pathogenic of PCV2b viruses is determined not in experimental infection.
Embodiment 2
The generation of PCV2b and chimeric PCV1-2b infectious DNA clone：
Have reported before infectious DNA clone for building PCV2a-40895 method (Fenaux, M. etc., 2002,
Ibid), PCV2b infectious DNA clone (Fig. 1 a) is produced using similar method in the present invention.In short, using one
To with comprising be present in it is all PCV2 plants in Sac II restriction enzyme sites overlay region primer A (SEQ ID No: 3)
With B (SEQ ID No:4) (table 1), PCV2b full-length genome is expanded by PCR.Then Sac II (New are used
England Biolabs) digest PCR primer and be connected to pBluescript II SK (+) (pBSK+) (Stratagene)
In to produce PCV2b infectious DNA clone.
In order to produce the infectious DNA clone of chimeric PCV1-2b, using Overlap extension PCR come by PCV1 infection clones
PCV1 capsid genes in skeleton are instead of the capsid gene (Fig. 1 b) from PCV2b.From 3 overlapping PCR fragments (table 1) assemblings
Total length is fitted together to PCV1-2b genomes.Using with identical Amplification (95 DEG C 3 minutes；40 circulation 95 DEG C 30 seconds, 55
DEG C 30 seconds, 68 DEG C 1 minute) Platinum Taq HiFi mastermix (Invitrogen) produce each amplicon.
Carry out purified pcr product using QIA quick gel extraction kits (QIAquick Gel Extraction Kit, Qiagen).
The fusion DNA vaccine being made up of two steps is used to assemble chimeric PCV1-2b DNA clones：Using 50ng each fragment as template not
Need primer assembly reaction (20 circulation 95 DEG C 30 seconds, 55 DEG C 30 seconds, 68 DEG C 1 minute), then using external primers
Amplification (40 circulation 95 DEG C 30 seconds, 55 DEG C 30 seconds, 68 DEG C 1 minute).Primer G (SEQ ID No will be used:And H 9)
(SEQ ID No:10) (table 1) from 1,043 bp fragments of PCV1 clonal expansions first with comprising with primer C (SEQ ID
No:5) with D (SEQ ID No:6) the 718 bp segment compositions of (table 1) from the PCV2b complete capsids genes expanded.Then
Add with primer E (SEQ ID No:7) with F (SEQ ID No:8) the 122 bp fragments that (table 1) is expanded from PCV1, are obtained
Flank is completely fitted together to PCV1-2b genomes for Kpn I restriction sites.Then with Kpn I (New England
Biolabs) digest chimeric fusion product and be cloned into pBSK+.Complete sequencing is carried out to monomer DNA clone to confirm in PCR
Without the unwanted mutation of introducing during amplification step.
Embodiment 3
The dimerization of PCV2b and PCV1-2b DNA clones：
Studies have shown that dimerization PCV2a before is cloned in both internal transfections of the in-vitro transfection of PK-15 cells and pig more
Effectively produce infectious virus, wherein the total length PCV2a genomes of 2 parts of copies by head and the tail series winding connecting (Fenaux,
M. etc., 2002, ibid；Fenaux, M. etc., 2004A, ibid；Fenaux, M. etc., 2003, ibid).Cause
This, in the present invention, clones both dimerizations to produce more healthy and stronger (robust) and effective infection by PCV2b and PCV1-2b
Property clone.In short, being extracted using QIAprep Spin Miniprep kits (Qiagen) comprising PCV2b and PCV1-2b
The DNA of monomeric gene group.Linearize the DNA of purifying using Sca I (New England Biolabs) and lead to
Cross 37 DEG C be incubated 30 seconds come be subjected to Kpn I it is partial digested with produce about 3,100 and 3,700 bp two fragments,
Then these fragments are purified by gel extraction.Two fragments are combined and connected using T4 DNA ligases (Promega)
With the infectious DNA clone of the series connection dimerization for producing both PCV2b and PCV1-2b.
Embodiment 4
Viability test of the infectious DNA clones of PCV2b and PCV1-2b in PK-15 cells：
As described above, using indirect fluorescence determination (IFA) come after transfecting, testing in vitro PCV2b and chimeric PCV1-2b DNA
The viability of clone and infectious (Fenaux, M. etc., 2002, ibid).In short, by 6.5 μ g each dimerization DNA
Clone is added in 1,250 μ l OPTIMEM culture mediums (Invitrogen) and 6.25 μ l PLUS reagents (Invitrogen), and
In incubation at room temperature 5 minutes.Add after 16 μ l Lipofectamine LTX (Invitrogen), by mixture in incubation at room temperature
30 minutes, add 250 μ l OPTIMEM culture mediums.The T25 Tissue Culture Flasks of the 60-70% PK-15 cells converged will be included
(Corning) washed with MEM culture mediums (Invitrogen), add transfection mixture, be then incubated bottle 6 hours at 37 DEG C.
After incubation, by the 8 ml growth mediums (MEM comprising 10% hyclone and 2x antibiotic/antimycotic solution
[Invitrogen]) add in each bottle, and it is incubated other 72 hours at 37 DEG C.Then transfectional cell in will be each T25 bottles-
80 DEG C of freezings and thaw 3 times, cell lysate is centrifuged 10 minutes to remove cell fragment at 4 DEG C with 2,500 x g.In harvest
Clear liquid simultaneously converges the fresh PK-15 cells for being inoculated in 48 orifice plates (BD-Falcon) with 50% for infecting.100 μ l are added per hole
Transfection supernatants after, plate is incubated 1 hour at 37 DEG C, backward each hole add 500 μ l growth mediums and in 37 DEG C of incubations
72 hours.As described above, capsid protein (Fenaux, M. in the core for the PK-15 cells that infection is visualized using IFA
Deng 2002, ibid).In short, washing 1 with PBS in 4 DEG C of fixed cells 30 minutes using 80% acetone in PBS
It is secondary, with 1:PCV2 monospecifics mouse monoclonal antibody (the Rural Technologies, Inc. of 1,000 dilutions；
Brookings, South Dakota) it is incubated 45 minutes at 37 DEG C.Washed with PBS after 3 times, cell is used 1:50 is dilute
The second goat anti-mouse IgG (KPL) for the FITC marks released is incubated 45 minutes at 37 DEG C.After being washed with PBS, then
Cell is covered with Fluoromount G (Southern Biotech) and is checked under fluorescence microscope.
When being transfected into PK-15 cells, PCV2b and chimeric PCVl-2b DNA clones are infective：PCV2b and embedding
The total length list copy and series connection dimerization DNA clone for closing PCV1-2b are fabricated and confirmation are sequenced by total length.With two kinds of DNA clones
Dimer transfection PK-15 cells cause the generation of infectious progeny virion, such as pass through IFA and PCV2 capsids specificity Dan Ke
Grand antibody is detected.The infectious titer of both viral reservoirs of PCV2b and chimeric PCV1-2b is about 104.5 TCID50/ml。
Embodiment 5
The generation and titration of the viral reservoir of infectious PCV2a, PCV2b and PCV1-2b：
In order to prepare the inoculum for pig In vivo study, the PK- in T25 bottles is transfected by using the infectious DNA clone of dimerization
15 cells (see above) (Fenaux, M. to produce PCV2a-40895, PCV2b and PCV1-2b infectious virus reservoir
Deng 2002. ibid；Fenaux, M. etc. 2003, ibid).Substantially carry out as described above, these are titrated by IFA
Infectious virus reservoir (Fenaux, M. etc. 2002, ibid).48 are inoculated in short, PK-15 cells are converged with 60%
Orifice plate (BD-Falcon), and be incubated 3 hours at 37 DEG C.10 times of dilutions of series of each viral reservoir are produced in MEM, and will
Each dilution is inoculated in four single holes with 100 μ l per hole.Plate is incubated 1 hour at 37 DEG C, backward each hole add 500
μ l growth mediums simultaneously continue to be incubated 72 hours at 37 DEG C.The positive letter of the core of infection cell in each hole is visualized using IFA
Number (seeing above).Every ml 50% tissue culture infection dose (TCID is calculated according to Reed ＆ Muench method50)。
Embodiment 6
The experiment that PCV2b and chimeric PCV1-2b deprives the Study on Pathogenicity in (CD/CD) pig of colostrum in caesarean birth source is set
Meter：
CD/CD pigs are considered as the excellent model system pathogenic for studying PCV2, because typical case can be reproduced in the model
Pathological lesion and clinic PCVAD (Allan, G. etc., 2003, ibid；Bolin, S. R. etc., 2001, ibid；
Harms, P. A. etc., 2001, ibid；Kennedy, S. etc., 2000, ibid；Tomas, A. etc., 2008, together
On).In order to determine the pathogenic of chimeric PCV1-2b viruses and be compared it with wild type PCV2b viruses, by about 9 week old
Amount to 30 CD/CD pigs (Struve Labs, Manning, IA) and be randomly assigned into three in the form of each 10 animals in room
Group.Before inoculation, each pig is weighed, bleeding and confirms that it exists for feminine gender to PCV2 antibody.1 pig of group is delayed with 3 ml PBS
Fliud flushing each simulates inoculation (2 ml are intranasal and 1 ml is intramuscular), and serves as the control being uninfected by.By 3 ml of pig organized in 2 bag
Containing 2 x 104.5TCID50The inoculum of chimeric PCV1-2b viruses is each inoculated with (2 ml are intranasal and 1 ml is intramuscular).By in group 3
Pig is with 2 x 104.5TCID50The each similar inoculation of wild type PCV2b viruses.Received before inoculation and after this from each pig weekly
Collect blood sample until the days post inoculation (dpi) of 21 or 42 days is autopsied.In 21 dpi, autopsy from each group 5
The pig being only randomly assigned.Remaining 5 pigs in 42 dpi, autopsy each group.
Embodiment 7
The experiment that PCV2b and chimeric PCV1-2b deprives the Study on Pathogenicity in (CD/CD) pig of colostrum in caesarean birth source is set
Meter：
CD/CD pigs are considered as the excellent model system pathogenic for studying PCV2, because typical case can be reproduced in the model
Pathological lesion and clinic PCVAD (Allan, G. etc., 2003, ibid；Bolin, S. R. etc., 2001, ibid；
Harms, P. A. etc., 2001, ibid；Kennedy, S. etc., 2000, ibid；Tomas, A. etc., 2008, together
On).In order to determine the pathogenic of chimeric PCV1-2b viruses and be compared it with wild type PCV2b viruses, by about 9 week old
30 CD/CD pigs (Struve Labs, Manning, IA) are amounted to be randomly assigned into the form of each 10 animals in room
Three groups.Before inoculation, each pig is weighed, bleeding and confirms that it exists for feminine gender to PCV2 antibody.1 pig will be organized with 3 ml PBS
Buffer solution each simulates inoculation (2 ml are intranasal and 1 ml is intramuscular), and serves as the control being uninfected by.By the pig organized in 2 with 3 ml's
Include 2 x 104.5TCID50The inoculum of chimeric PCV1-2b viruses is each inoculated with (2 ml are intranasal and 1 ml is intramuscular).It will organize in 3
Pig with 2 x 104.5TCID50The each similar inoculation of wild type PCV2b viruses.Before inoculation and after this weekly from each pig
Blood sample is collected until the days post inoculation (dpi) of 21 or 42 days is autopsied.In 21 dpi, it is autopsied from each group
5 pigs for being randomly assigned.Remaining 5 pigs in 42 dpi, autopsy each group.
Chimeric PCVl-2b viruses are attenuation in CD/CD pigs, and wild type PCV2b virus inductions PCVAD pathology is damaged
Evil and clinical disease characteristic：In order to clearly assess the pathogenic potential of chimeric PCV1-2b viruses, caused in CD/CD pig models
Characteristic of disease study, the model have been shown as most reproducible and super-sensitive PCVAD disease models (Allan, G. etc., 2003,
Ibid；Bolin, S. R. etc., 2001, ibid；Harms, P. A. etc., 200,1 ibid；Kennedy, S. etc.
2000, ibid).
Clinical sign and gross lesion ：Run through with the CD/CD pigs of chimeric PCV1-2b viruses or PBS experiment inoculation
No PCVAD obvious clinical sign is entirely studied, and is infected with wild type PCV2b experiment inoculation CD/CD pigs in 10 PCV2b
4 of pig in cause the related death of PCVAD or early stage to be euthanized：1 pig is dead in 18 dpi, and another is dead in 27 dpi
Die, remaining is two only as progressive weight loss has to be euthanized in 34 dpi.
The pig of each from 3 treatment groups is until 21 dpi have similar increased weight, but wild type PCV2b
Have in 4 after 21 dpi in 5 pigs of the pig of infection increased weight decline (because 3 pigs of weight loss are dead or
It is euthanized in early days).Do not observe macroscopic lung damage in the pig with chimeric PCV1-2b virus inoculations, and 27
The pig of 3 PCV2b inoculations of dpi and 34 dpi autopsys shows medium lung damage.Compared with PBS control, PCV1-2b and
Lymph node enlargement in the pig of both PCV2b infection.However, there is the pig quantity for expanding lymph node in the pig of PCV1-2b infection
With extensive magnitude than few (data are not shown) in wild type PCV2b pigs.
Micro- infringement ：Micro- infringement is analyzed by the processing in following two groups：21 dpi or before be autopsied
All pigs (pig for being included in the dead PCV2b infection of 18 dpi), and (it is included in 27 in the 42 dpi all pigs being autopsied
Dpi and 34 dpi death or the pig for the 3 PCV2b infection being euthanized).
Micro- infringement in lung, liver, thymus gland, the heart, kidney, ileum, colon, lymph node, spleen and tonsillotome is summarized in table 2.
As expected, without significant micro- infringement in the pig being inoculated with PBS.In 21 and 42 dpi, and with wild type
The pig of PCV2b experiment inoculations is compared, aobvious with the typical PCV2 correlations in the lymphoid tissue of the pig of chimeric PCV1-2b virus inoculations
The incidence and seriousness of micro- infringement all decline (table 2).Micro- infringement in other non-lymphoid tissues includes lymphocyte and huge
Phagocyte it is slight to severe infiltration (table 2).Small intestine and large intestine are almost exclusively found that in the pig that wild type PCV2b infects
In infringement.
In 21 dpi, with the overall lymph infringement scoring of the average value group of the pig of chimeric PCV1-2b virus inoculations significantly (p=
0.045) it is less than the scoring with the pigs of wild type PCV2b virus inoculations, and is had no difference with the pig that is inoculated with PBS
(Fig. 3).
PCV2 serology ：By the blood serum sample obtained from pig that is dead or being euthanized in early days with making a reservation for from next step
The blood serum sample of autopsy is analyzed (for example, including coming comfortable 18 in the sample that analysis is autopsied from 21 dpi together
The serum of pig dead dpi).It is special that just PCV2 capsids are detected early in 7 dpi in the serum of some PCV1-2b pigs infected
Property IgG antibody, wherein 9/10 pig to 21 dpi seroconversions (Fig. 2).PCV2 in the pig of chimeric PCV1-2b virus inoculations
IgG antibody potency reaches that stable and until research, which terminates (42 dpi), keeps high to 28 dpi.Viral with wild type PCV2
In the pig for testing inoculation, seroconversion is not relatively consistent：Only 3/10 pig is to 21 dpi seroconversions, and 5/10 pig is in the research
In do not have detectable seroconversion.However, the pig of only 2/5 PCV2b infection lives to dpi 42 predetermined autopsy, and
Both carry out seroconversion in 28 dpi, until research terminates all have increased IgG antibody potency.Due to clinical PCVAD
In death or the pig being euthanized in early days, only 1/4 has detectable PCV2 specific antibodies at any time, without pig at it
That day for being autopsied there is the anti-PCV2 antibody of detectable serum.It is inoculated with through the research in any PBS
PCV2 capsid specific antibodies are all not detected by pig.
The amount of illness rate and PCV2 specific antigens in the tissue determined by IHC ：Illness rate in different tissues and
The average value group amount of PCV2 antigens is summarized in table 3.Analyze together 21 dpi or before all pigs for being autopsied (be included in
The pig of PCV2b infection dead 18 dpi), and analyze together be autopsied in 42 dpi all pigs (be included in 27 dpi and
34 dpi death or the pig for the 3 PCV2b infection being euthanized).Generally, compared with the pig of wild type PCV2b virus inoculations,
It is all relatively low (table 3) with the incidence of disease of the pig of chimeric PCV1-2b virus inoculations and the amount of PCV2 antigens.Except the external institute of almond
There is the pig for having 1 PCV1-2b inoculation in 21 dpi tissues to cause positive findings, 4 in tonsillotome in 5 pigs are the positive.
42 dpi that are organized in from the PCV1-2b pigs being inoculated with are largely feminine gender, wherein except lymph node, 5 pigs in lymph node
In 4 only detect a small amount of PCV2 antigens.
PCV2 viremia virusemias and serum viral load ：Determine using the improvement qPCR of amplification part PCV2b capsid genes
PCV2 virus loads in the serum for the animal for determining infection, it is known that qPCR is determined and worked to both PCV2b and PCV1-2b
(Yang, Z. Z. etc., 2007, Detection of PCV2 DNA by SYBR Green l-based
Quantitative PCR (PCV2 DNA are detected by the quantitative PCR based on the green l of SYBR) J Zhejiang Univ Sci
B 8:162-9).Confirm before inoculation 0 day and the pig that is inoculated with through the research from PBS in all blood serum samples for obtaining
Without detectable PCV2 DNA.The serum that will be obtained from pig that is dead due to PCVAD diseases or being euthanized in early days
Sample is analyzed together with the blood serum sample from the predetermined autopsy of next step (for example, when analyzing the sample from 21 dpi
Serum including carrying out the dead pigs of comfortable 18 dpi).
From 14 dpi up to research terminates, serum viral load present in the pig with chimeric PCV1-2b virus inoculations shows
Write the serum viral load (Fig. 4 a) in the pig that (p≤0.009) is inoculated with less than PCV2b.Average blood in the pig of PCV1-2b infection
Clear virus load reaches peak value 10 in 14 dpi7Genome copies/ml, and steadily decline until research terminates.For any list
The maximum that the pig of individual PCV1-2b infection is realized is the 10 of 14 dpi8Genome copies/ml.In contrast to this, PCV2b feels
Average value serum viral load in the pig of dye is from 14 dpi until being all kept above 10 after 28 dpi8Genome copies/ml, its
In the maximum realized of single pig be 1012.Due to PCVAD, Deaths or 4 pigs being euthanized connect than other PCV2b
The pig planted has the serum viral load of higher level.After 7 dpi, the viral genome of every ml serum of the pig of PCV2b infection
Copy is at least 10 times of the pig of PCV1-2b infection, and is 100 times in 21 dpi and 28 dpi.
PCV2 virus loads in lymphoid tissue ：Determine to determine in each pig that is autopsied using identical qPCR such as above
When collected TBLN tissues in PCV2 viral DNAs amount.Confirm all TBLN tissues of pig being inoculated with from PBS
Sample is feminine gender to PCV2 DNA.In order to the meaningful ratio for the cell mean virus load for carrying out 21 dpi and 42 dpi
Compared with, test but in analysis not include due to PCVAD diseases Deaths or the pig being euthanized.It was found that in 21 dpi
Virus load present in the TBLN tissues of every mg in the pig of PCV1-2b inoculations is substantially less than the disease in the pig of PCV2b inoculations
Malicious carrying capacity (p=0.005) (Fig. 4 b).It is dead due to PCVAD or be euthanized in early days in all tested TBLN tissues
Pig TBLN tissue in all have highest virus load.
Embodiment 8
Chimeric PCV1-2b viruses are in routine without the vaccine inoculation in specific pathogen (SPF) pig and the reality of immunogenicity research
Test design：
It is miscellaneous that 40 3 week old are amounted to from commercial farm (Virginia Tech Swine Center, Blacksburg, VA) purchase
Hand over kind of a SPF pigs, it is known that these pigs are free of PCV, PRUSV, PPV, mycoplasma hyopneumoniae and pig hepatitis E virus.Piggy is random
2 groups are distributed into, every group of 20 pigs are simultaneously housed individually.Before inoculation, each pig is weighed, bleeding and confirm its to PCV2 antibody for the moon
Property.Chimeric PCV1-2b virus respective intramuscular (IM) vaccine inoculation (every pig 10 of 1 pig with 1 ml will be organized3.5 TCID50It is infectious
Virus).2 pigs will be organized, and with 1 ml PBSs, each IM simulates vaccine inoculation and serves as the control of non-vaccine inoculation.Per heavenly prison or jail
Control the clinical sign of all animals, and before inoculation and after this until number of days (dpv) is weekly after the vaccine inoculation of 56 days
Blood sample is collected from each pig.
Embodiment 9
Attack and intersect the experimental design for the pig for attacking vaccine inoculation respectively with wild type PCV2b hypotypes and PCV2a hypotypes：
In 56 dpv, the pig of 20 vaccine inoculations is further divided into two groups of every group of 10 pigs, and in single room centre circle
Support：The pig of 10 vaccine inoculations is each with 2 x 104.5 TCID50(2 ml are intranasal and 1 ml for wild type PCV2b virus attacks
IM), it is the pig of other 10 vaccine inoculations is each with 2 x 104.5 TCID50Intersect attack as wild type PCV2a virus types.Also
The control pig of 20 non-vaccine inoculations is further divided into two groups, every group of 10 pigs, and respectively with PCV2b and PCV2a similarly
Inoculation.Blood sample is collected weekly up to number of days (dpc) (or 77 dpv) after the attack of 21 days, at that time by all pig corpse
Dissection.
Assigned with chimeric PCV1-2b viral vaccinations for the attack of PCV2b hypotypes and the intersection attack of PCV2a hypotypes conventional
The protective immunity of pig：Because the field that chimeric PCV1-2b vaccines are intended to conventional pig is used, inventor uses routine SPF
Pig carries out the efficacy of vaccines and Attack Research.
Clinical sign and gross lesion ：There is no the pig clinical sign consistent with PCVAD through the research, and in processing
The difference of increased weight is not observed between group.Be assigned to 1 pig of non-vaccine inoculation/PCV2b attack groups 2 dpc die from
The incoherent bacterial septicemias of PCVAD, and be not included in analysis.What in autopsy, all do not seen in treatment group in office
Observe macroscopic lung damage.The slight lymph node to medium expansion is all seen in all treatment groups, wherein handling it
Between do not observe significant difference.
Serology ：It is feminine gender to the antibody for PCV2, PRRSV, PPV and SIV that all pigs are determined before research.Early in 14
Dpv just detects PCV2 capsid specific antibodies in the serum of the pig of PCV1-2b vaccine inoculations, wherein in 21 dpv 15/
Seroconversion occurs for 20 pig, and occurs seroconversion (Fig. 5) to 28 dpv 20/20 pig.PCV2 IgG antibodies potency is to 35
Dpv reaches stable and keeps high when 56 dpv are attacked.Until after 14 dpc attacks, in the control pig of non-vaccine inoculation
It is not detected by anti-PCV2 antibody (Fig. 5)：In 14 dpc, compared with the pig that the PCV2b of only 2/9 seropositivity is attacked, 10/10
The pig of PCV2a attacks is seropositivity.To 21 dpc, the Swine serum transformation of the PCV2b attacks of 7/9 non-vaccine inoculation.
Micro- infringement ：After attack, compared with the pig of vaccine inoculation, the micro- damage in the lymphoid tissue of the pig of non-vaccine inoculation
The incidence and seriousness of evil scoring are higher (table 4).Generally, compared with the control of non-vaccine inoculation, the pig of vaccine inoculation
Relatively low (table 4) is scored in typical lymph atrophy and histocyte displacement.For PCV2a (p=0.0001) and PCV2b (p=0.04)
Both attack groups, the overall lymph infringement scoring of the pig of vaccine inoculation is substantially lower than the scoring (Fig. 6) of the pig of non-vaccine inoculation.
The amount of PCV2 antigens in the tissue determined by IHC ：IHC is used to detect in each organization type (table 4)
PCV2 specific antigens.It is similar with histologic lesion, compared with the control of non-vaccine inoculation, the generally tissue of the pig of vaccine inoculation
In incidence and the amounts of PCV2 antigens all reduce (table 4).
Viremia virusemia and serum viral load ：Determined using improvement qPCR come PCV2a after quantitative attack in serum or
The amount of PCV2b viral DNAs, the measure is to detection PCV2a or PCV2b rep gene specifics but can not expand vaccine virus
PCV1-2b (the Development and validation of a SYBR green of Mcintosh, K. A. etc. 2009.
real-time PCR for the quantification of porcine circovirus type 2 in serum,
Buffy coat, feces, and multiple tissues (are developed and verified for quantitative serum, buffycoat, excreta
With the green real-time PCR of SYBR of 2 type pig circular ring virus in Various Tissues) Vet Microbiol 133:23-33).Confirm
The blood serum sample collected before 56 dpv attacks does not have detectable PCV2 DNA.Any time point after attack is in inoculation
There is no detectable PCV2a or PCV2b viremia virusemias in the pig of vaccine.In contrast to this, each time point after attack is in institute
Have and PCV2a or PCV2b viremia virusemias (Fig. 7 a) are all detected in the pig of non-vaccine inoculation.With PCV2a or PCV2b virus attacks
Non- vaccine inoculation pig between in terms of serum viral load without statistically significant difference.
PCV2a or PCV2b virus loads in lymphoid tissue ：During autopsy PCV2a collected in TBLN tissues or
The amount of PCV2b viral DNAs is summarized in fig .7b.In PCV2a (p=0.0001) and PCV2b (p<0.0001) both attack groups
In, compared with the pig of non-vaccine inoculation, it is found that the virus load in the TBLN tissues in the pig of vaccine inoculation per mg is significantly lower
(Fig. 7 b).The pig of only 1/10 vaccine inoculation and PCV2b attacks has detectable PCV2b viral DNAs in TBLN tissues,
Wherein virus load is 105Genome copies/mg.The pig of 5/10 vaccine inoculation and PCV2a attacks has in TBLN tissues
There are detectable PCV2a viral DNAs, wherein respective virus load is 105-106Genome copies/mg.The pig of non-vaccine inoculation
TBLN tissue in virus load scope be 108-1010Genome copies/mg, are attacked but regardless of PCV2a or PCV2b.
Experiment material and method
Cell：The PK-15 cell lines of no PCV1 pollutions are produced by end dilution PK-15 cells (ATCC CCL-33) before
Subclone (Fenaux, M. etc., 2000, ibid；Fenaux, M. etc., 2002, ibid).By the PK-15 without PCV1
Cell line is used for the infectious titration for producing the viral reservoir that infectious virus reservoir and the present invention are used.
Serology：As described above, ELISA is used to detect the anti-PCV2 IgG in each blood serum sample
(Nawagitgul, P. etc., 2002, Modified indirect porcine circovirus (PCV) type 2-
based and recombinant capsid protein (ORF2)-based enzyme-linked immunosorbent
Assays for detection of antibodies to PCV (be used for detect anti-PCV antibody improvement it is indirect
Enzyme linked immunosorbent assay (ELISA) based on 2 type pig circular ring virus (PCV) and based on recombinant capsid protein (ORF2)), Clin Diagn
Lab Immunol 9:33-40).Blood serum sample has sample：The positive (S:P) it is considered as confrontation PCV2 antibody sun than being more than 0.2
Property.It is PCV2 seronegativities to confirm all pigs by ELISA before zoopery is started.
Clinical evaluation：After inoculation, vaccine inoculation or attack, the PCVAD of pig clinical sign is evaluated daily, PCVAD's faces
Bed body is levied including becoming thin, respiratory distress and Behavioral change (such as drowsiness and poor appetite).
Visually visible pathology and histopathology：At the appointed time carrying out autopsy to all pigs in blind mode is used for
Pathogenic (dpi 21 and 42) or attack experiment (dpc 21 or dpv 77).Carry out macroscopic lung damage (the lung model of infection
Enclose for 0-100%) and lymph nodes size (scope be 0 [normal] -3 [4 times of normal sizes]) estimation, and to the scoring of each pig
(Halbur, P. G. etc., 1995, Comparison of the pathogenicity of two US porcine
reproductive and respiratory syndrome virus isolates with that of the
Lelystad virus (the pathogenic ratios of two kinds of US porcine reproductive and respiratory syndrome virus coniviums and lelystad virus
Compared with), Vet Pathol 32:648-60；Opriessnig, T. etc., 2004, ibid).
Lung, lymph node (groin surface, vertical diaphragm, tracheobronchial and intestines system are collected during each autopsy
Film), tonsillotome, the heart, thymus gland, ileum, kidney, colon, the section of spleen and liver, and be fixed in 10% neutral buffered formalin
It is used for histological examination and immunohistochemistry (IHC) with conventional treatment.Equally, tracheobronchial lymph nodes is collected from each pig
(TBLN) sample is used for DNA extractions and by real-time PCR come quantitative viral genome.As described above, with blind mode to lung,
Micro- infringement scoring (Opriessnig, T. etc., 2004, ibid) in the heart, liver, kidney, ileum and colon.Withered based on lymph
The histocyte displacement of contracting and folliculus have rated lymphoid tissue including lymph node, spleen and tonsillotome, and scope is 0 (just
Often) -3 (serious) (Opriessnig, T. etc., 2004, ibid).
Immunohistochemistry (IHC)：Lung, the lymph node of FFPE are fixed to formalin using rabbit polyclonal antiserum
(groin surface, vertical diaphragm, tracheobronchial and mesenteric mesaraic), tonsillotome, the heart, thymus gland, ileum, kidney, colon, spleen and
The section of liver carries out IHC (the Development of of Sorden, S. D. etc. 1999. for being used to detect PCV2 specific antigens
a polyclonal-antibody-based immunohistochemical method for the detection of
Type 2 porcine circovirus in formalin-fixed, paraffin-embedded tissue (exploitation bases
It is used for the 2 type pig annulus for detecting that formalin is fixed in the tissue of FFPE in the immunohistochemical method of polyclonal antibody
Virus) J Vet Diagn Invest 11:528-30).The scoring of the PCV2 antigens in each tissue is estimated in blind mode,
Scoring scope be 0 (nonantigenic) -3 (cell that contains the dyeing of positive PCV2 antigens more than 50% lymph follicle in lymphoid tissue or
PCV2 amount of antigen in other histotomies of person is high) (Opriessnig, T. etc., 2004, ibid).
Overall micro- lymph infringement scoring：As described above, the average score of overall micro- lymph infringement is calculated each pig
(Opriessnig, T. etc., 2004, ibid).These infringement scorings are based on the lymph combined present in lymphoid tissue
Atrophy (LD), histocyte displacement (HR) and PCV2 antigens, as determined by IHC.
Quantitative real-time PCR determines viral DNA carrying capacity：According to " the blood and body fluid " scheme provided by manufacturer, use
QIAamp DNA minikit (Qiagen Inc) extract STb gene from blood serum sample.Will be collected during autopsy
TBLN organizes homogenization to produce 10% tissue homogenate suspension in sterile PBS buffer, and according to manufacturer (Qiagen
Inc) " tissue " scheme provided, STb gene is extracted using QIAamp DNA minikit.All TBLN DNA extracts are existed
At least with 1 in sterilized water:100 dilutions, to eliminate from the background fluorescence green SYBR combined with pig genomic DNA.Due to
Viral DNA concentration extremely high in some samples, some serum and TBLN extracts are diluted up to 1:106, so as to make they
In the range of linearity of qPCR detections.Two kinds of qPCR based on the green I of SYBR are determined into improvement is used for the present invention, with quantitative TBLN and blood
PCV2 genomes (Mcintosh, K. A. etc., 2009, Development and present in clear DNA extracts
validation of a SYBR green real-time PCR for the quantification of porcine
In serum, buffy coat, feces, the and multiple tissues of circovirus type 2 (are developed and tested
Demonstrate,prove the green real-time PCR of SYBR for 2 type pig circular ring virus in quantitative serum, buffycoat, excreta and Various Tissues), Vet
Microbiol 133:23-33；Yang, Z. Z. etc., 2007, ibid).
In CD/CD pig Study on Pathogenicity, inventor is determined using a kind of qPCR announced before, measure amplification part
PCV2b capsid genes are with viral genome present in quantitative serum and TBLN (Yang, Z. Z. etc., 2007, ibid).
Modification enables to use Sensimix SYBR and luciferase reagent box (Quantace).Each 25 μ l are anti-
Each primers of 200 nM (P1 (SEQ ID No should be included: 11): 5'-ATAACCCAGCCCTTCTCCTACC-3')；P2 (SEQ
ID No: 12): 5'-GGCCTACGTGGTCTACATTTCC-3')、200 µM dNTP、3mM MgCl2Extracted with 5 μ l DNA
Thing.Use the program (95 DEG C 10 minutes of modification；35 circulation 95 DEG C 15 seconds, 59 DEG C 30 seconds, 72 DEG C 30 seconds), in MyIQ
Triplicate reaction is run in qPCR temperature cyclings device (BioRad) to each sample.Use relative CtMethod, infects for PCV2b
Property DNA clone the quantitative viral genome of repetition standard items, quantitative (10 with the 6-log ranges of linearity3-108Copy).
In attack and intersection attack routine SPF pig researchs, inventor is determined using a kind of qPCR of announcement before, the survey
Surely the high conservative region in amplification PCV2 rdrp genes area is with virus genomic amount present in quantitative serum and TBLN tissues
(Mcintosh, K. A. etc., 2009, ibid).The measure does not detect the chimeric PCV1-2b epidemic diseases used in vaccine inoculation
Seedling diseases poison, and therefore allow only accurate quantification attack PCV2a or PCV2b viruses.The modification program enables to use
Sensimix SYBR and luciferase reagent box.Each 25 μ l reactions include each primers of 200 nM (PCV2-83F (SEQ ID No:
13): 5'-AAAAGCAAATGGGCTGCTAA-3'；PCV2-83R (SEQ ID No: 14): 5'-
TGGTAACCATCCCACCACTT-3')、200 µM dNTP、5mM MgCl2With 5 μ l DNA extracts.Use the program of modification
(95 DEG C 10 minutes；35 circulation 95 DEG C 15 seconds, 60 DEG C 15 seconds, 72 DEG C 15 seconds), in MyIQ qPCR temperature cycling devices
Triplicate reaction is run to each sample.Use relative CtMethod, determines for the repetition standard items of the infectious DNA clones of PCV2b
Viral DNA genome is measured, the quantitative (5x10 with the 5-log ranges of linearity2-5x106Copy).
Confirmed by the qPCR viral sequences detected：Test the TBLN tissue DNAs extracted from the selected pig of each group
Extract, with confirm by qPCR detect it is viral with each research when the virus that is inoculated into pig it is identical.Using to PCV1-2b
Special primer (PCV2F (SEQ ID No: 15): 5'-TGTTGAAGATGCCATTTTTCC-3'；PCV1R (SEQ ID
No: 16):5'-GAGGAGTTCTACCCTCTTCC-3'), ground by PCR amplifications and part sequencing to confirm that CD/CD is pathogenic
PCV1-2b in studying carefully.Using to primer (PCV2F (SEQ ID No special PCV2: 17): 5'-
TGTTGAAGATGCCATTTTTCC-3'；PCV2R (SEQ ID No: 18):5'-GAGGTGTTCGTCCTTCCTCA-3'),
Amplification and sequencing PCV2a and PCV2b.
Statistical analysis：Serology and serum qPCR are analyzed using measure variance analysis (ANOVA) is repeated.Using simple
ANOVA analyzes the TBLN qPCR from CD/CD Study on Pathogenicity.For all ANOVA models, Glimmix programs are used
Slice options compare to study simple effect, are used for Multiple range test using Tukey program afterwards.Use accurate Wilcoxon
The TBLN qPCR results from Attack Research are analyzed in 2 sample tests.Assessed with the unidirectional ANOVA of accurate Kruskal-Wallis
The scoring of lymph infringement, histopathologic lesions and IHC from two groups of experiments, afterwards by the accurate sample tests of Wilcoxon 2
For two-way comparison of interest.Two-way compare for Multiple range test is adjusted using Bonferroni program.Statistics is aobvious
Work property is set to α=0.05.All analyses are carried out using commercial software (SAS versions 9.2, Cary, NC, USA).
Inactivated vaccine Suvaxyn PCV2 have been developed based on the chimeric prepared from PCV2a hypotypes before inventor®
One dose™(Fenaux, M. etc. 2004, ibid；Fenaux, M. etc. 2003, ibid), the killed vaccine is effective
And can be obtained on world market at present.By using similarly tactful, in our current research, inventor develops a kind of new first
Embedded virus PCV1-2b, the capsid gene of wherein PCV2b hypotypes is cloned into non-pathogenic PCV1 genome skeleton.In CD/CD
New the one of attenuation living is used as with fully have studied using the chimeric PCV1-2b viruses for PCV2 and PCVAD in conventional pig model
For vaccine.
For using chimeric PCV1-2b viruses as attenuated vaccine living, it is important to determine vaccine virus is attenuation.Though
Right PCV2 individually seldom causes the full spectrum clinical disease in experimental model, but using CD/CD pigs caused clinical PCVAD into
Work(reproduction (Allan, G. etc., 2003, ibid；Bolin, S. R. etc., 2001, ibid；Harms, P. A. etc.,
2001, ibid；Kennedy, S. etc., 2000, ibid；Tomas, A. etc., 2008, ibid).Therefore, in the present invention
In, in order to clearly determine the pathogenic of chimeric PCV1-2b viruses and be compared it with its parental wild-type PCV2b viruses, invent
People's selection CD/CD pig models are used for Study on Pathogenicity.As expected, wild type PCV2b individually causes the severe of CD/CD pigs to be faced
Bed PCVAD, causes the pig that 4/10 PCV2b infect dead due to PCVAD clinical manifestation or early stage euthanasia.This with before
Display PCV2 infection CD/CD pigs 25% death rate it is consistent with significant clinic PCVAD research (Allan, G. etc.,
2003, ibid；Bolin, S. R. etc., 2001, ibid；Harms, P. A. etc., 2001, ibid).PCV2b infects
Pig overall lymph infringement scoring with it is medium to the systemic PCVAD of severe it is consistent (Opriessnig, T. etc. 2007, together
On).Although host's variation prevents individually clearly diagnosis cutoff (cut-off), the viremia virusemia of severe PCVAD cases
General acceptable thresholds be per ml serum 107Viral genome copies (Brunborg, I. M. etc., 2004,
Quantitation of porcine circovirus type 2 isolated from serum/plasma and
tissue samples of healthy pigs and pigs with postweaning muitisystemic
Wasting syndrome using a TaqMan-based real-time PCR (using the real-time PCR based on TaqMan come
Quantitative 2 types separated from health pig and in the serum/plasma and tissue sample of the pig with postweaning multisystem exhaustion syndrome
Pig circular ring virus), J Virol Methods 122:171-8；Olvera, A. etc., 2004, Comparison of
porcine circovirus type 2 load in serum quantified by a real time PCR in
postweaning muitisystemic wasting syndrome and porcine dermatitis and
Nephropathy syndrome naturally affected pigs are (by the postweaning multisystem exhaustion of Real-Time PCR quantitation
The comparison of 2 type pig circular ring virus carrying capacity in the serum of the pig of syndrome and the scorching nephrotic syndrome natural infection of pigskin), J Virol
Methods 117:75-80；Segales, J. etc., 2005, Quantification of porcine circovirus
type 2 (PCV2) DNA in serum and tonsillar, nasal, tracheo¬bronchial, urinary
and faecal swabs of pigs with and without postweaning muitisystemic wasting
Syndrome (the PMWS) (serum of the quantitative pig for suffering from and not suffering from postweaning multisystem exhaustion syndrome (PMWS) and flat
2 type pig circular ring virus (PCV2) DNA in peach body, nose, trachea-bronchia, the assay specimen of urine and excrement), Vet
Microbiol 111:223-9).To 21 dpi, compared with the pig of only 1/10 chimeric PCV1-2b virus infection, 9/10
The pig of PCV2b infection exceedes the threshold value.It is dead due to PCVAD or by 4 pigs of relatively early euthanasia all in serum and lymph group
There is higher PCV2 DNA virus carrying capacity in knitting, but without detectable PCV2 IgG antibodies, this shows that these PCV2b feel
The pig of dye dies from the immune response of deficiency and the virus replication of higher level.The result and the low antibody response reported before and increasing
Plus PCVAD seriousness between correlation consistent (Meerts, P. etc., 2006, Correlation between the
presence of neutralizing antibody against porcine circovirus 2 (PCV2) and
protection against replication of the virus and development of PCV2-
(there is the neutralizing antibody for 2 type pig circular ring virus (PCV2) with being directed to virus replication and generation in associated disease
Correlation between the protection of PCV2 relevant diseases), BMC Vet Res 2:6).Data clearly show what is used in the present invention
PCV2b hypotypes are high virulence.
Although being inoculated with CD/CD pigs with the dosage of at least 20 times normal vaccination doses, it was found that chimeric PCV1-2b
Virus is (Fenaux, the M. etc., 2004, ibid) being significantly attenuated in CD/CD pig models.Chimeric PCV1-2b viruses
It is attenuated and understands proof by all qualitatively and quantitatively parameters for relatively more chimeric PCV1-2b and wild type PCV2b viruses.With about one
The death seen in the pig of half PCV2b infection and becoming thin is compared, and death is not seen in the pig infected with chimeric PCV1-2b viruses
Or morbidity.The amount of micro- infringement scoring and PCV2 specific antigens in the lymphoid tissue of the pig of chimeric PCV1-2b infection is notable
Less than the pig infected with wild type PCV2b, show that chimeric PCV1-2b viruses only cause subclinical infection.Generally, it is fitted together to
PCV1-2b infection pig lymph infringement scoring be inoculated with PBS compare pig without dramatically different.In addition, serum and
Significantly lower typical lesions or disease in the pig that relatively low PCV2b virus loads infect with chimeric PCV1-2b in lymphoid tissue
Seriousness is directly related, observed by studying as in the previous (Brunborg, I. M. etc., 2004, ibid；Dupont,
K. etc., 2009, Transmission of different variants of PCV2 and viral dynamics in
a research facility with pigs mingled from PMWS-affected herds and non-
Affected herds (the biographies of the different PCV2 variants in the research facilities that the swinery of PMWS infection mixes from the swinery being uninfected by
Pass and dynamics of virus), Vet Microbiol 139:219-26；Fenaux, M. etc., 2004, ibid；Harding,
J. C etc., 2008, Porcine circovirus-2 DNA concentration distinguishes wasting
from nonwasting pigs and is correlated with lesion distribution, severity,
And nucleocapsid staining intensity (DNA concentration of pig circular ring virus -2 distinguishes become thin pig and the non-pig that becomes thin,
And related to infringement distribution, seriousness and nucleocapsid staining power), J Vet Diagn invest 20:274-82；
Krakowka, S. etc., 2005, Features of porcine circovirus-2 disease: correlations
Between lesions, amount and distribution of virus, and clinical outcome (justify by pig
The feature of the disease of circovirus virus -2：Infringement, the amount of virus and distribution and the correlation of clinical effectiveness), J Vet Diagn Invest
17:213-22；Mcintosh, K. A. etc., 2009, ibid；Olvera, A. etc., 2004, ibid；Yang, Z. Z.
Deng, 2007, ibid).
After proving chimeric PCV1-2b viruses in susceptible and sensitiveness CD/CD pig models for attenuation, inventor
Then combination immunogenicity and Attack Research have been carried out in conventional SPF pigs.3 week old conventional hybridization kind SPF pigs are selected to be used to exempt from
Epidemic focus/attack experiment more to simulate field vaccine inoculation condition closely, because such attenuated vaccine living will finally be used
In conventional pig.Although the research more early announced based on us, be not expected in the conventional SPF models clinic PCVAD (Fenaux,
M. etc., 2002, ibid；Fenaux, M. etc., 2004, ibid；Fenaux, M. etc., 2004, ibid；Fenaux,
M. etc., 2003, ibid, Fenaux, M. etc., 2004A. is ibid；Opriessnig, T, etc., 2009,
Difference in severity of porcine circovirus type two-induced pathological
(it is tight that the pathology of 2 type pig circular ring virus induction are damaged lesions between Landrace and Pietrain pigs
Difference of the principal characteristic between Land race and Pietrain pigs), J Anim Sci 87:1582-90； Opriessnig, T.
Deng, 2008, ibid), it is contemplated that by the viremia virusemia in the lymphoid tissue of the PCV2 conventional pig models induced, virus load
With Representative histology damage level be for evaluate the abundant parameter of efficacy of vaccines (Fenaux, M. etc., 2004, together
On).It is used as attenuated vaccine living, it is important to determine whether chimeric PCV1-2b viruses can induce enough when to pig vaccine inoculation
The protection antibody reaction of level.2 kinds in 4 kinds of current available vaccines are to be based on restructuring PCV2a capsid proteins, therefore known
The reaction of PCV2 capsids specific humoral immunity is important to protecting.The result of the present invention is shown, early in 14 dpv just in PCV1-2b epidemic diseases
Detect PCV2 capsid specific antibodies in the serum of the pig of seedling inoculation, and to 20 all 28 dpv vaccine inoculations pig all
Seroconversion is anti-PCV2 capsids specific antibody.Antibody titer reaches stable and keeps high when 56 dpv are attacked to 35 dpv
, show that chimeric PCV1-2b vaccine viruses can cause the strong humoral immune reaction in conventional pig.
When being attacked with wild type PCV2a or PCV2b hypotype, compared with the control of non-vaccine inoculation, with being fitted together to for attenuation
The conventional SPF pigs of PCV1-2b viral vaccinations have significantly lower viral DNA carrying capacity in serum and TBLN tissues, shown
PCV2 specificity in the seriousness and incidence level and significantly lower lymphoid tissue of the typical micro- infringement for writing reduction is anti-
Former amount, shows that chimeric PCV1-2b virus inductions are directed to the protective immunity of Wild-Type Virus Challenge.As a result also do not show, and not
The control of vaccine inoculation is compared, with the pigs of chimeric PCV1-2b viral vaccinations for the homologous challenge of PCV2b hypotypes and
The Heterologous Challenge of PCV2a hypotypes is thus equally protected, and this is by completely without in PCV2a or PCV2b viremia virusemias, lymphoid tissue
Significantly lower overall lymph infringement in the pig substantially reduced with vaccine inoculation of viral 7 carrying capacity is scored and confirmed, but regardless of
Challenge virus hypotype.Therefore, it appears that guarantor of the new work attenuated chimeric PCV1-2b vaccine candidate objects induction for two kinds of PCV2 hypotypes
Shield property is immunized and cross-protective immunity.
Some nearest researchs are it has been reported that when compared with PCV2a hypotypes, usual PCV2b hypotypes and more serious clinic
Disease correlation (Carman, S. etc., 2008, ibid；Chae, J. S. and K. S. Choi, 2009, ibid；
Grau-Roma, L etc., 2008, A proposal on porcine circovirus type 2 (PCV2)
genotype definition and their relation with postweaning muitisystemic wasting
Syndrome (PMWS) occurrence is (to 2 type pig circular ring virus (PCV2) VDA genotypes and its and postweaning multisystem
The suggestion for the relation that exhaustion syndrome (PMWS) occurs), Vet Microbiol 128:23-35).But, if PCV2b is sub-
Type more has virulence still disputable than PCV2a hypotype, because other researchs can not be explicitly shown PCV2a and PCV2b in virulence
Aspect significant difference (An, D. J. etc., 2007, ibid；Lager, K. M. etc., 2007, ibid；Madson,
D. M. etc., 2008, ibid；Opreissnig, T. etc., 2006, ibid；Opreissnig, T. etc., 2008,
Ibid).Due to the sequence divergence between PCV2a and PCV2b, PCV2 two kinds of hypotypes can not be both possible at pathogenic aspect
Because it has been shown that in cap genes the change of only two amino acid be enough to change PCV2a it is pathogenic (Fenaux, M. etc.,
2004, Two amino acid mutations in the capsid protein of type 2 porcine
circovirus (PCV2) enhanced PCV2 replication in vitro and attenuated the virus
(2 amino acid mutations in the capsid protein of 2 type pig circular ring virus (PCV2) strengthen external PCV2 to be replicated and internal in vivo
Make virus attenuation), J Viol 78:13440-6).In the control group of the non-vaccine inoculation of current research, the wherein pig of half
Attacked with PCV2a or PCV2b, inventor does not observe any significant difference in terms of virulence between group.PCV2a and PCV2b
Challenge dose be equivalent, but serum and PCV2 virus loads in TBLN tissues, typical micro- infringement scoring or drench
The amount of PCV2 antigens in bar tissue is between PCV2a and PCV2b attack groups without significant difference.In the pig of non-vaccine inoculation,
Compared with PCV2a, there are some less differences, including the seroconversion (Fig. 5) slightly postponed to PCV2b antibody response.Connecing
In the pig for planting vaccine, PCV2b attacks cause slightly higher overall lymph to damage scoring, and PCV2a attacks cause comparatively high amts
TBLN tissues positive qPCR.Generally, it is as a result pathogenic under experimental conditions without significantly with other discovery PCV2a and PCV2b
The research of difference it is consistent (An, D. J. etc., 2007, ibid；Lager, K. M etc., 2007, ibid；Madson, D.
M. etc., 2008, ibid；Opriessnig, T. etc., 2006, ibid；Opriessnig, T. etc., 2008, together
On).
Have reported the difference of the antigen overview between PCV2a and PCV2b hypotypes, thus it is speculated that be currently based on PCV2a business
Enough cross protection level attributables that vaccine is lacked PCVAD seriousness related to PCV2b hypotypes in global swinery
Increase (Cheung, A. K. etc., 2007, ibid；Dupont, K. etc., 2008, ibid；Lekcharoensuk,
P. etc., 2004, ibid；Shang, S. B. etc., 2009, ibid).Most sequence variations between PCV2a and PCV2b
Seem in capsid gene (including the unique amino acid motif of signal) (Cheung, A. K. etc., 2007, ibid；
Olvera, A. etc., 2004, ibid).The antibody produced for the motif can distinguish PCV2a and PCV2b in vitro, show
Antigenic possible different (Beach etc., data are not announced).The present invention result show, the capsid based on new PCV2b hypotypes
Work attenuated vaccine PCV1-2b really for PCV2a Heterologous Challenge protect, therefore support before the inactivation business based on PCV2a
The cross protection for the PCV2b hypotypes that industry vaccine is assigned evidence (Fort, M., etc., 2008, ibid；Fort, M. etc.,
2009, ibid；Opriessnig, T. etc., 2009, ibid；Opriessnig, T. etc., 2008, ibid).
In a word, as shown by data of the invention, the chimeric PCV1-2b vaccine candidate objects based on new PCV2b hypotypes are in CD/CD
It is attenuation in pig, and for PCV2b and PCV2a attacks inducing protective immunity and friendship in the conventional pig of vaccine inoculation respectively
Pitch protective immunity.The result of the present invention will be felt further to develop the chimeric PCV1-2b viruses as first for PCV2
Dye and PCVAD work attenuated vaccine provide basis.Although PCV1-2b vaccine viruses are provided separately for PCV2a and PCV2b hypotypes
Cross protection, but the PCV1-2b vaccines of the combination present invention and be currently based on PCV2a commercial vaccine and be used for more complete protect
Shield can be in the future more favourable.
Vaccine and be also included in the scope of the present invention using their method that infectious virus and molecular dna are cloned
It is interior.The pig of inoculation is protected against serious virus and infected and the other diseases as caused by PCV2 infection or coinfection.New side
Method is by giving the vaccine of the present invention of immune effective dose to pig (for example, the infectious PCV DNA comprising immunogenicity amount, containing
The vaccine of the plasmid or viral vector of PCV infectious DNA clone, restructuring PCV DNA, Polypeptide expression products etc.) it is directed to disease
Poison infection protection needs pig to be protected.Other antigens (such as PRRSV, PPV), other infectious swine diseases can be given simultaneously to pig former
Body and immunologic stimulant are with the protection for virus infection offer wide spectrum.
Vaccine is included, for example with nontoxic physiologically acceptable carrier and choose any one kind of them or a variety of adjuvant combinations infection
Venereal disease poison and molecular dna are cloned, the PCV infectivity DNA genomes of suitable plasmids or the clone in carrier (such as pSCK carriers),
Nontoxic live virus, virus of inactivation etc..Vaccine can also include infectious PCV2 molecular dnas clone as described herein.It is infectious
PCV DNA, the DNA comprising infectious virus genome and live virus are preferred, and wherein live virus is most preferred.This
The nontoxic live-virus vaccine of invention provides advantage relative to traditional viral vaccine, and traditional viral vaccine is using returning to poisonous shape
The live attenuated virus of state risk or the intact virus of dead cell culture breeding, it may not induce enough antibody mediated immunities anti-
Protected applied to for virus disease.
Vaccine and also it is included within the scope of the invention using their method.The mammalian species of inoculation, which are protected, to be exempted from
In serious virus infection, protection can be also provided the disease for being related to PCV coinfections, the disease such as PCVAD and pigskin are scorching
Nephrotic syndrome (PDNS) and other relevant diseases.Vaccine includes viral, the nontoxic lifes of such as inactivation or attenuation pig TTV
Acceptable carrier and chosen any one kind of them or a variety of adjuvants in reason.
Adjuvant is the material for increasing pig to the immune response of vaccine, and it can jointly give with the vaccine of the present invention.Can be with epidemic disease
Seedling on identical site or in different time (for example, being used as booster immunization) while and give adjuvant.Also can by adjuvant with
The mode different from the mode for giving vaccine advantageously gives pig on the different site in the site with giving vaccine.Suitably
Adjuvant includes but is not limited to aluminium hydroxide (alum), immunostimulating complex (ISCOMS), non-ionic block polymers or common
Polymers, cell factor (as IL-1, IL-2, IL-7, IFN-α, IFN-β, IFN-γ etc.), saponin(e, monophosphoryl lipid A (MLA),
Muramyl dipeptide (MDP) etc..Other suitable adjuvants include, such as aluminum potassium sulfate, be isolated from Escherichia coli (Escherichia coli) thermally labile or heat-staple enterotoxin, cholera toxin or its B subunits, diphtheria toxin, tetanus toxin, pertussis
Toxin, Freund are not exclusively or Freund's complete adjuvant etc..Can be by adjuvant (such as diphtheria toxin, tetanus toxin and one hundred days based on toxin
Cough toxin) inactivate before the use, such as by using formaldehyde treated.
Vaccine can further include other antigen to promote the immunocompetence of infectious PCV DNA clones, the antigen
Such as porcine reproductive and respiratory syndrome virus (PRRSV), pig parvoviral (PPV), other infectious porcine pathogens and immune thorn
Swash thing.
The novel vaccine of the present invention is not limited to any particular type or preparation method.The viral vaccine of clone includes but is not limited to
Infectious DNA vaccination (carrying out direct injection DNA into pig using plasmid, carrier or other conventional carriers), live vaccine, improvement
Live vaccine, inactivated vaccine, subunit vaccine, attenuated vaccine, recombinant vaccine etc..These vaccines are by standard side known in the art
It is prepared by method.
As other benefit, preferred live vaccine of the invention provides a kind of vaccine of inheritance stability, and it compares other types
Attenuated vaccine be easier to prepare, preserve and deliver.
Another preferred vaccine of the present invention delivers nonpathogenic DNA clone using suitable plasmid into pig.With using
Live virus or the traditional vaccine of intact virus of dead cell culture breeding are compared, and the present invention is provided with including venereal infection
The direct Pigs Inoculated of DNA of virus gene group.
The other recombinant vaccine that institute's phase of the present invention needs is produced by techniques known in the art.Such technology includes
But it is not limited to other operation, the modification of the amino acid sequence of recombinant protein or displacement of recombinant DNA etc..
Recombinant vaccine based on recombinant DNA technology is, for example, to be responsible for inducing relatively strong immune in pig by identification code
The alternative part of the viral gene of the albumen (albumen such as from ORF3, ORF4) of reaction or aversion response is made
It is standby.The gene or immunodominant fragments so identified can be cloned into standard protein expression vector (such as baculovirus vector)
In, and for infect suitable host cell (see, for example, O'Reilly etc., " Baculovirus Expression
Vectors:A Lab Manual (rhabdovirus expression vectors：Laboratory manual), " Freeman ＆Co., 1992).Host
Cell is cultured, therefore the vaccine protein needed for expression, and vaccine protein can be purified to required degree and be configured to suitably
Vaccine product.
If clone retains any undesirable instinct for causing disease, then find out in viral genome and take responsibility
The nucleotide sequence of what residual virulence and to transform viral gene nontoxic as example, by direct mutagenesis be also possible.It is fixed
Point mutagenesis can increase, lack or change one or more nucleotides (see, for example, Zoller etc., DNA 3:479-488,
1984).Oligonucleotides of the synthesis comprising required mutation and the part for being annealed into single-stranded viral DNA.By what is obtained from the step
Hybrid molecule is used to convert bacterium.Then, separation is used for by with being then transfected into conjunction comprising the double-stranded DNA being properly mutated
The restriction fragment of full length DNA in suitable cell culture connects to produce full length DNA.Genome is connected to for shifting
Suitable carrier in can be realized by any standard technique known to those of ordinary skill in the art.Any routine side can be used
Carrier is transfected into the host cell for producing daughter of virus by method, the conventional method such as calcium phosphate or deae dextran
Transfection, electroporation, protoplast fusion and other known technologies of mediation are (for example, Sambrook etc., " Molecular
Cloning:A Laboratory Manual (molecular clonings：Laboratory manual), " Cold Spring Harbor
Laboratory Press, 1989).The virus of clone then shows required mutation.Alternatively, it can synthesize comprising suitable prominent
Two oligonucleotides become.These nucleotides can anneal can be plugged into viral DNA to produce the double-strand of full length DNA to be formed
DNA。
The vaccine of the present invention of immune effective dose, which is given, needs the pig for virus infection protection.It can be held by routine test
Easily determine or easily titrate the immune effective dose or immunogenicity amount of Pigs Inoculated.Effective dose is to realize enough exempting from for vaccine
Epidemic disease reaction is exposed to the amount of the viral pigs of PCV to protect.Preferably, pig is protected to a kind of to whole bad physiology of virus disease
The degree that symptom or effect are significantly reduced, mitigate or prevented completely.
Vaccine can be given with single dose or repeated doses.Dosage range can be the bag of the microgram of e.g., from about 1 microgram-about 1,000
DNA (concentration for depending on the immune active ingredient of vaccine) containing infectious chimeric DNA genome, preferably 100-200
The chimeric PCV1-2 DNA clones of microgram, but the base for the physiological signs for being enough to cause adverse reaction or virus infection should not be included
In the amount of antigen of virus.For determining or titrate the suitable dose of active antigenic substance to find the body weight based on pig, antigen
The method of the minimum effective dose of concentration and other typical factors is known in the art.Preferably, infectious virus DNA clone
It is used as vaccine, or infectious virus living can be produced in vitro and the live virus is then used as vaccine.In such case
Under, for example can be by the about 50- about 10,000 of live virus 50% tissue culture infection dose (TCID50) give pig.
The novel vaccine of the present invention is not limited to any particular type or preparation method.Vaccine includes but is not limited to the work epidemic disease of improvement
Seedling, inactivated vaccine, subunit vaccine, attenuated vaccine, recombinant vaccine etc..
What the advantage of live vaccine was to activate vaccine recipient is possible to immune response, including whole body, part, body fluid and carefully
The immune response of born of the same parents' mediation.The shortcoming of live-virus vaccine is to have can by the possibility or virus of adventitious viruses Substances Pollution living
The risk of virulence is replied in the wild, and the shortcoming can exceed that advantage.
In order to prepare the viral vaccine of inactivation, for example, can culture pig cell system (such as, but not limited to PK-15 cells)
Middle generation virus breeding and virus production.Then by the generally known scheme of those of ordinary skill in the art or preferably by this
Method described in text optimizes continuous inactivation of virus.
Can by using inactivator (such as formalin or hydrophobic solvent, acid), by using ultraviolet or X-ray radiation,
The viral vaccine of inactivation is prepared by the processing pig circular ring virus such as heating.Inactivated in the way of this area understands.For example,
In chemical ablation, by the appropriate virus sample comprising virus or the inactivator of blood serum sample sufficient amount or concentration sufficiently high
The sufficiently long time is handled with inactivation of viruses at the temperature or pH of (or low, depending on inactivator).It is in foot by heat inactivation
To be carried out under the temperature and time length of inactivation of viruses.It is light or other energy source spokes using certain wavelength by radiological inactivation
Penetrate and be enough the time span of inactivation of viruses to carry out.If virus can not infect the cell of susceptible, then the virus is recognized
For be inactivation.
Also it is that desirable recombinant vaccine is produced by techniques known in the art in the present invention.Such technology bag
Include but be not limited to using RNA, recombinant DNA, recombinant protein, live virus etc..
, can be by methods known in the art from suitable clinical, biological sample (such as serum, row for example, after purification
Let out thing, saliva, seminal fluid and tissue sample) in separation wild-type virus, the preferred method by instructing herein uses the pig of infection
Or the appropriate cell line of infection.DNA is extracted from the pure virus of biology or infectious agent by methods known in the art, and passes through ability
Method known to domain is purified, and preferably passes through CsCl gradient ultracentrifugations.By methods known in the art by viral base
Because group cDNA clone into suitable host (referring to Maniatis etc., id.), then analyze viral genome with determine use
In the essential regions for the genome for producing viral antigen part.Thereafter, step is generally with improveing live vaccine, inactivated vaccine or subunit
The step of vaccine, is identical.
For example, the albumen for the stronger immune response or aversion response for being responsible for inducing pig by identification code (is such as immunized
Virus protein, such as derives from ORF2 capsid protein) the part of viral gene prepare the gene based on recombinant DNA technology
Engineered vaccine.The gene or immunodominant fragments of this identification can be cloned into standard protein expression vector (such as baculoviral
Carrier) in, and for infecting suitable host cell (see, for example, O'Reilly etc., " Baculovirus
Expression Vectors:A Lab Manual (rhabdovirus expression vectors：Laboratory manual), " Freeman ＆Co.,
(1992)).Host cell is cultured, therefore the vaccine protein needed for expression, and vaccine protein can be purified to required degree simultaneously
It is configured to suitable vaccine product.
Alternatively, the DNA for the pig PCV from separation that can will encode one or more capsid proteins inserts carrier living
In (such as poxvirus or adenovirus) and as vaccine.
The vaccine of the present invention of immune effective dose, which is given, needs pig or lactation for the infection or syndrome protection to move
Thing species.It can easily be determined by routine test or easily titrate " immune effective dose ".Effective dose is to realize enough to be directed to epidemic disease
The immune response of seedling with protect be exposed to pig TTV virus pig or other mammals amount, pig TTV viruses can cause PCVAD,
The disease of pigskin inflammation nephrotic syndrome (PDNS) or correlation.Preferably, pig or other mammalian species are protected to virus disease
A kind of to whole bad physiological signs or effect be found the degree that significantly reduces, mitigate or prevent completely.
Vaccine can be given with single dose or repeated doses.Dosage can be included, for example 1-1, the resisting based on virus of 000 microgram
Former (concentration for depending on the immunoactive component of vaccine), but the life for being enough to cause adverse reaction or virus infection should not be included
Manage the amount of antigen based on virus of symptom.The method of suitable dose for determining or titrating active antigenic substance be this area
Know, its body weight based on birds or mammal, the concentration of antigen and other typical factors.
Pig can be given by vaccine.Equally, vaccine can be given to people (such as pig farm that excessive risk is infected by viral agent
It is main).Orally available, cheek is interior, intranasal, percutaneous, parenteral etc. eligibly to give vaccine.It is parenteral to give approach including but not limited to flesh
Interior, intravenous, intraperitoneal and subcutaneous route.
When being given as liquid, this vaccine can be prepared in the form of aqueous solution agent, syrup, elixir, tincture etc..
Such preparation is known in the art and by the way that antigen and other typical additives are dissolved in into suitable carrier or solvent
Typically prepared in system.Suitable carrier or solvent include but is not limited to water, salt solution, ethanol, ethylene glycol, glycerine etc..Allusion quotation
The additive of type is, such as certification dyestuff (certified dye), flavorant, sweetener and antimicrobial preservative (such as sulphur
Willow mercury (second mercury salicylate sulfonium sodium)).Can be for example by adding the gelatin, D-sorbite or cell culture medium of partial hydrolysis come steady
Fixed such solution, and reagent known in the art (such as disodium hydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, phosphorus can be used
Acid dihydride potassium, their mixture etc.), such solution is buffered by conventional method.
Liquid preparation also may include supensoid agent and emulsion, and it includes what is combined with other standards auxiliary agent (coformulant)
Suspending agent or emulsifying agent.The liquid preparation of these types can be prepared by conventional method.Colloid mill for example can be used in supensoid agent
To prepare.For example homogenizer can be used to prepare for emulsion.
Designed for being expelled to the suitable isotonicty of parenteral formulation needs of humoral system and being buffered to mammalian body
The pH value of the respective horizontal of liquid.Optionally isotonicty can be suitably adjusted with sodium chloride and other salt.Suitable solvent is (for example
Ethanol or propane diols) it can be used for increasing the solubility and the stability of liquid preparation of the composition in preparation.It can make in this vaccine
Other additive includes but is not limited to glucose, Conventional antioxidants and conventional cheating agents (such as ethylenediamine tetra-acetic acid)
(EDTA).Also must be sterile before parenteral dosage form use.
Claims (15)
1. a kind of viral vaccine, it is selected from following member comprising physiologically acceptable carrier and immunogenicity amount：
(a) PCV1-2b viruses are fitted together to, it is obtained by the nucleic acid molecules for being fitted together to PCV1-2b, wherein PCV1 ORF2 capsid genes
Replaced by the ORF2 capsid genes of wild type PCV2b strains subtypes；
(b) the chimeric PCV1-2b viruses of the capsid protein comprising PCV2b strains；With,
At least one other pig antigen of optional immunogenicity amount.
2. the viral vaccine of claim 1, wherein the vaccine be vaccine living, the vaccine of the work of modification, the vaccine of inactivation or
The vaccine of attenuation.
3. the viral vaccine of claim 2, wherein the vaccine is the vaccine of inactivation.
4. the viral vaccine of claim 2, wherein the vaccine is vaccine living.
5. the viral vaccine of claim 1, wherein the vaccine is for PCV2a and PCV2b infection protections.
6. the viral vaccine of claim 1, wherein pig antigen optionally, in addition is infectious porcine pathogen.
7. the viral vaccine of claim 1, breeds with exhaling wherein pig antigen optionally, in addition is selected from chimeric PCV1-2a viruses, pig
Inhale syndrome virus (PRRSV), pig parvoviral (PPV) and its combination.
8. the viral vaccine of claim 1, wherein pig antigen optionally, in addition is inactivation, chimeric PCV1-2a viruses.
9. the viral vaccine of claim 1, it additionally comprises adjuvant.
10. any one of claim 1-9 viral vaccine is being prepared in the vaccine for the viral infection immunity pigs of PCV2
Purposes.
11. the purposes of claim 10, wherein the vaccine prepared is used for single dose or repeated doses are administered.
12. the purposes of claim 11, wherein the vaccine prepared is administered for single dose.
13. the purposes of claim 10, wherein the vaccine prepared is used for parenteral, intranasal, intracutaneous or percutaneous dosing.
14. the purposes of claim 10, wherein the vaccine prepared is used in lymph or intramuscular administration.
15. any one of claim 1-9 viral vaccine is being prepared for for the related disease (PCVAD) of pig circular ring virus
Protect the purposes in the vaccine of pig.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US31436210P | 2010-03-16 | 2010-03-16 | |
| US61/314362 | 2010-03-16 | ||
| CN2011800244674A CN102971425A (en) | 2010-03-16 | 2011-03-16 | Live attenuated chimeric porcine circovirus vaccine |
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| US7279166B2 (en) * | 2001-12-12 | 2007-10-09 | Virginia Tech Intellectual Properties, Inc. | Chimeric infectious DNA clones, chimeric porcine circoviruses and uses thereof |
| US7833707B2 (en) | 2004-12-30 | 2010-11-16 | Boehringer Ingelheim Vetmedica, Inc. | Methods of overexpression and recovery of porcine circovirus type 2 ORF2 |
| UA95602C2 (en) | 2004-12-30 | 2011-08-25 | Берингер Ингельхейм Ветмедика, Инк. | Pcv2 immunogenic compositions and methods of producing such compositions |
| MY149398A (en) | 2005-12-29 | 2013-08-30 | Boehringer Ingelheim Vetmed | Multivalent pcv2 immunogenic compositions and methods of producing such compositions |
| MX2008008311A (en) | 2005-12-29 | 2009-03-04 | Boehringer Ingelheim Vetmed | Use of a pcv2 immunogenic composition for lessening clinical symptoms in pigs. |
| EP2101815A4 (en) | 2006-12-11 | 2010-10-06 | Boehringer Ingelheim Vetmed | Effective method of treatment of porcine circovirus and lawsonia intracellularis infections |
| US8865183B2 (en) | 2006-12-15 | 2014-10-21 | Boehringer Ingelheim Vetmedica, Inc. | Treatment of pigs with PCV2 antigent |
| EP1941903A1 (en) | 2007-01-03 | 2008-07-09 | Boehringer Ingelheim Vetmedica Gmbh | Prophylaxis and treatment of PRDC |
| EP1958644A1 (en) | 2007-02-13 | 2008-08-20 | Boehringer Ingelheim Vetmedica Gmbh | Prevention and treatment of sub-clinical pcvd |
| US7829274B2 (en) | 2007-09-04 | 2010-11-09 | Boehringer Ingelheim Vetmedica, Inc. | Reduction of concomitant infections in pigs by the use of PCV2 antigen |
| KR20100113582A (en) | 2008-01-23 | 2010-10-21 | 베링거잉겔하임베트메디카인코퍼레이티드 | Pcv2 mycoplasma hyopneumoniae immunogenic compositions and methods of producing such compositions |
| CN102971425A (en) * | 2010-03-16 | 2013-03-13 | 弗吉尼亚科技知识产权有限公司 | Live attenuated chimeric porcine circovirus vaccine |
| WO2015026912A1 (en) * | 2013-08-23 | 2015-02-26 | Boehringer Ingelheim Vetmedica, Inc. | Porcine circovirus type 2 (pcv2) subunit vaccine |
| JP6821429B2 (en) | 2013-09-25 | 2021-01-27 | ゾエティス・サービシーズ・エルエルシー | PCV2B branched vaccine composition and usage |
| US9505808B2 (en) | 2013-10-02 | 2016-11-29 | Boehringer Ingelheim Vetmedica, Inc. | PCV2 ORF2 protein variant and virus like particles composed thereof |
| US10555994B2 (en) * | 2015-03-30 | 2020-02-11 | Boehringer Ingelheim Animal Health USA Inc. | PCV2 ORF2 carrier platform |
| CN104984335A (en) * | 2015-07-14 | 2015-10-21 | 浙江诺倍威生物技术有限公司 | Construction of PCV (Porcine Circovirus) double subtype ORF2 co-expression vector and vaccine preparation |
| CN108431024A (en) * | 2015-10-16 | 2018-08-21 | 堪萨斯州立大学研究基金会 | 3 type circovirus immunogenic composition of pig and its preparation and application |
| WO2017187277A2 (en) * | 2016-03-07 | 2017-11-02 | Virginia Tech Intellectual Properties, Inc. | Chimeric porcine circovirus type 2 (pcv2) vaccines |
| CN111892659A (en) * | 2020-07-20 | 2020-11-06 | 武汉科前生物股份有限公司 | Chimeric porcine circovirus PCV1-2d vaccine and preparation method and application thereof |
| CN114908065B (en) * | 2022-05-07 | 2023-10-31 | 中国农业大学 | Porcine pseudorabies virus genetic engineering attenuated vaccine strain, and establishment method and application thereof |
Citations (2)
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| CN1693455A (en) * | 2005-04-08 | 2005-11-09 | 扬州大学 | Mosaic type swine circular ring virus PCV 1.2 and its construction method use |
| US20090017064A1 (en) * | 2007-07-10 | 2009-01-15 | Wyeth | Methods and Compositions for Immunizing Pigs Against Porcine Circovirus |
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| US7276353B2 (en) | 2001-12-12 | 2007-10-02 | Virginia Tech Intellectual Properties, Inc. | Chimeric infectious DNA clones, chimeric porcine circoviruses and uses thereof |
| US7279166B2 (en) | 2001-12-12 | 2007-10-09 | Virginia Tech Intellectual Properties, Inc. | Chimeric infectious DNA clones, chimeric porcine circoviruses and uses thereof |
| TW200643171A (en) * | 2005-06-07 | 2006-12-16 | Temasek Life Sciences Lab Ltd | Porcine circovirus type 2 vaccines |
| KR20100094587A (en) | 2007-12-21 | 2010-08-26 | 와이어쓰 엘엘씨 | Methods and compositions for immunizing pigs against porcine circovirus |
| CN102971425A (en) * | 2010-03-16 | 2013-03-13 | 弗吉尼亚科技知识产权有限公司 | Live attenuated chimeric porcine circovirus vaccine |
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| CN1693455A (en) * | 2005-04-08 | 2005-11-09 | 扬州大学 | Mosaic type swine circular ring virus PCV 1.2 and its construction method use |
| US20090017064A1 (en) * | 2007-07-10 | 2009-01-15 | Wyeth | Methods and Compositions for Immunizing Pigs Against Porcine Circovirus |
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| BR112012023234A2 (en) | 2016-05-17 |
| HRP20200258T1 (en) | 2020-09-04 |
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| EP2547770A4 (en) | 2013-10-16 |
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| US20110280905A1 (en) | 2011-11-17 |
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| SI2547770T1 (en) | 2020-07-31 |
| PL2547770T3 (en) | 2020-06-29 |
| US20170173143A1 (en) | 2017-06-22 |
| PT2547770T (en) | 2020-03-04 |
| HUE047972T2 (en) | 2020-05-28 |
| EP3639852A1 (en) | 2020-04-22 |
| CY1122684T1 (en) | 2021-03-12 |
| US20140220067A1 (en) | 2014-08-07 |
| US9585951B2 (en) | 2017-03-07 |
| CN102971425A (en) | 2013-03-13 |
| WO2011116094A2 (en) | 2011-09-22 |
| ES2773722T3 (en) | 2020-07-14 |
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